Nucleic acids for inhibiting expression of tmprss6 and iron chelators

ABSTRACT

The present invention relates to products and compositions and their uses. In particular the invention relates to nucleic acid products that interfere with the TMPRSS6 gene expression or inhibits its expression in combination with one or more iron chelators and possibly other active agents, as well as therapeutic uses such as for the treatment of hemochromatosis, porphyria and blood disorders such as beta-thalassemia, sickle cell disease and transfusional iron overload or myelodysplastic syndrome, and infections and non-relapse related mortality associated with bone marrow transplantation.

The present invention relates to products and compositions and theiruses. In particular the invention relates to nucleic acid products thatinterfere with the TMPRSS6 gene expression or inhibits its expression,in combination with other active agents, and therapeutic uses such asfor the treatment of hemochromatosis, porphyria and blood disorders suchas β-thalassemia, sickle cell disease and transfusional iron overload,and infections and non-relapse related mortality associated with bonemarrow transplantation.

BACKGROUND

Double-stranded RNA (dsRNA) able to complementarily bind expressed mRNAhas been shown to be able to block gene expression (Fire et al, 1998,Nature. 1998 Feb. 19; 391(6669):806-11 and Elbashir et al., 2001,Nature. 2001 May 24; 411(6836):494-8) by a mechanism that has beentermed RNA interference (RNAi). Short dsRNAs direct gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and have become a useful tool for studying gene function. RNAi ismediated by the RNA-induced silencing complex (RISC), asequence-specific, multi-component nuclease that destroys messenger RNAshomologous to the silencing trigger loaded into the RISC complex.Interfering RNA (iRNA) such as siRNAs, antisense RNA, and micro-RNA areoligonucleotides that prevent the formation of proteins bygene-silencing i.e. inhibiting gene translation of the protein throughdegradation of mRNA molecules. Gene-silencing agents are becomingincreasingly important for therapeutic applications in medicine.

According to Watts and Corey in the Journal of Pathology (2012; Vol 226,p 365-379) there are algorithms that can be used to design nucleic acidsilencing triggers, but all of these have severe limitations. It maytake various experimental methods to identify potent siRNAs, asalgorithms do not take into account factors such as tertiary structureof the target mRNA or the involvement of RNA binding proteins. Thereforethe discovery of a potent nucleic acid silencing trigger with minimaloff-target effects is a complex process. For the pharmaceuticaldevelopment of these highly charged molecules it is necessary that theycan be synthesised economically, distributed to target tissues, entercells and function within acceptable limits of toxicity.

Matriptase-2 (MT-2) is the product of the TMPRSS6 gene. MT-2 is a typeII transmembrane serine protease that plays a critical role in theregulation of iron homeostasis. MT-2 is a negative regulator for geneexpression of the peptide hormone hepcidin. Hepcidin is predominantlyproduced and secreted by the liver. Hepcidin regulates iron balance inthe body by acting as a negative regulator of gastro-intestinal ironabsorption and release of iron from cellular stores. Upregulation ofhepcidin leads to a reduction in iron availability within the body(McDonald et al, American Journal of Physiology, 2015, vol 08 no. 7,C539-C547). Hepcidin levels are low in patients with iron overload.Therefore a possible target for reducing iron overload is to increaseHepcidin by silencing TMPRSS6 gene expression in the liver.

TMPRSS6 is primarily expressed in the liver, although high levels ofTMPRSS6 mRNA are also found in the kidney, with lower levels in theuterus and much smaller amounts detected in many other tissues (Ramsayet al, Haematologica (2009), 94(*6), 84-849).

Various disorders are associated with iron overload, a conditioncharacterised by increased blood and tissue iron levels, such ashereditary hemochromatosis, porphyria cutanea tarda, and blooddisorders, like β-thalassemia, congenital sideroblastic anemia (CSA),congenital dyserythropoietic anemia (CDA), marrow failure syndromes,myelodysplasia and sickle cell disease (SCD). In addition all patientpopulations, that receive regular blood transfusions are at risk ofdeveloping transfusional iron overload (Coates, Free Rad Biol Med 2014,Vol 72, 23-40, Bulaj et al., Blood 2000, Vol 95, 1565-71). Both a paperby Nai et al (Blood, 2012, Vol 119, No. 21, p 5021-5027) and Guo et al(The Journal of Clinical Investigation, 2013, Vol 123, No. 4, p1531-1541) show how a deletion or reduction of TMPRSS6 expression waseffective in treating β-thalassemia in mice. A paper by Schmidt (2013,Blood, Vol 121, 7, p 1200-1208) explores the use of TMPRSS6 nucleic acidto decrease iron overload in mouse models of hereditary hemochromatosisand β-thalassemia. In both models the authors determined that lipidnanoparticles—TMPRSS6 nucleic acid treatment induced hepcidin anddiminished tissue and serum iron levels for up to 21 days. Furthermore apaper by Schmidt et al (American Journal of Hematology, 2015, Vol 90,No. 4, p 310-313) demonstrated that LNP-TMPRSS6 nucleic acid plus oraliron chelator deferiprone therapy reduced secondary iron overload inmouse model of β-thalassemia. Further, there is evidence that patientswith iron overload that undergo bone marrow transplantation experiencean enhanced risk for infection and non-relapse related mortality.Enhanced systemic iron levels increase the risk of infections due toimpairment of immunity and by heightened microbial virulence. Patientsreceiving bone marrow transplantations are particularly vulnerable toinfections as the cytotoxic conditioning required to prepare thepatients for transplantation ablates their bone marrow and increasesalso systemic iron levels as erythropoiesis is impaired. Furthermore,high systemic iron levels may have a negative impact on the implantationand outgrowth of the grafted donor stem cells.

Reducing iron overload and systemic iron levels before and duringconditioning of patients receiving bone marrow transplantation mayreduce the risk of infections and non-relapse related mortality (Wermkeet al., 2018, Lancet Haematol. May; 5 (5):e201-e210).

Accordingly, methods for effective treatment of disorders associatedwith iron overload are currently needed and the present inventionaddresses this need.

The invention relates to a nucleic acid or conjugated nucleic acidaccording to any aspect of the disclosure, in combination with one ormore iron chelators.

Nucleic Acids and Conjugated Nucleic Acids

A first aspect disclosed herein relates to a nucleic acid for inhibitingexpression of TMPRSS6, comprising at least one duplex region thatcomprises at least a portion of a first strand and at least a portion ofa second strand that is at least partially complementary to the firststrand, wherein said first strand is at least partially complementary toat least a portion of a RNA transcribed from the TMPRSS6 gene, whereinsaid first strand comprises a nucleotide sequence selected from thefollowing sequences: SEQ ID NOs: 317, 319, 321, 323, 325, 327, 329, 331,333, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394,396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422,424, 426, 428, 430, 432, 434, 436, 438, 440, or 442, or the first strandcomprises a sequence selected from SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 67, 69, 71, 73, 74, 76, 77, 78, 79, 80, 81,82, 83, 85, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 270, 271, 273, 275, 277, 279, 281, 283,285, 287, 289, 291, 293, 295, 297, 299, 301, 309, 311, 312, 313, 314, or315, such as SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or215.

A further related aspect is a nucleic acid for inhibiting expression ofTMPRSS6, comprising at least one duplex region that comprises at least aportion of a first strand and at least a portion of a second strand thatis at least partially complementary to the first strand, wherein saidfirst strand is at least partially complementary to at least a portionof a RNA transcribed from the TMPRSS6 gene, wherein said second strandcomprises a nucleotide sequence selected from the following sequences:SEQ ID NOs 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,342, 344, 346, 357, 359. 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, or 443, or the second strand comprises a nucleotidesequence selected from the following sequences: SEQ ID NOs 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 68, 70, 72, 75, 84, 86, 89,100, 101, 102, 103, 104, 105, 106, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 226, 228, 230, 232, 234, 236, 238, 240,242, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 310, 312, 314, or 316, such as a sequence selected from thefollowing: SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or216.

Certain nucleic acids are preferred in all of the aspects of theinvention. In particular: The first strand may comprise the nucleotidesequence of SEQ ID NO:333 and/or the second strand ma comprise thenucleotide sequence of SEQ ID NO:334.

333 TMPRSS6-hcm-9A aaccagaagaagcagguga 334 TMPRSS6-hcm-9Bucaccugcuucuucugguu

The first strand may comprise the nucleotide sequence of SEQ ID NO:17and/or the second strand may comprise the nucleotide sequence of SEQ IDNO:18, namely:

SEQ ID 17 TMPRSS6-hcm-9A 6273646282647284546 SEQ ID 18 TMPRSS6-hcm-9B1727354715351718451

The first strand may comprise the nucleotide sequence of SEQ ID NO:422and/or the second strand may comprise the nucleotide sequence of SEQ IDNO:423.

TMPRSS6-hc-18A UUUUCUCUUGGAGUCCUCA TMPRSS6-hc-18B UGAGGACUCCAAGAGAAAA

The first strand may comprise the nucleotide sequence of SEQ ID NO:199and/or the second strand may comprise the nucleotide sequence of SEQ IDNO:200.

TMPRSS6-hc-18A mU (ps) fU (ps) mUfUmCfUmCfUmUfGmGfAmGfUmCfCmU (ps)fC (ps) mA TMPRSS6-hc-18BfUmGfAmGfGmAfCmUfCmCfAmAfGmAfGmAfA (ps) mA (ps) fA

The first strand may comprise the nucleotide sequence of SEQ ID NO:429and/or the second strand may comprise the nucleotide sequence of SEQ IDNO:430.

TMPRSS6-hc-23A CUGUUCUGGAUCGUCCACU TMPRSS6-hc-23B AGUGGACGAUCCAGAACAG

The first strand may comprise the nucleotide sequence of SEQ ID NO:207and/or the second strand may comprise the nucleotide sequence of SEQ IDNO:208.

TMPRSS6-hc-23A mC (ps) fU (ps) mGfUmUfCmUfGmGfAmUfCmGfUmCfCmA (ps)fC (ps) mU TMPRSS6-hc-23BfAmGfUmGfGmAfCmGfAmUfCmCfAmGfAmAfC (ps) mA (ps) fG

A nucleic acid may be disclosed herein in a sequence listing associationwith a particular linker and/or ligand, but any reference to the linkeror ligand in the context of the sequence listing is optional, althoughthese features are preferred. Certain preferred linkers are disclosed incombination with certain nucleic acid sequences.

The first strand and/or said second strand may each be from 17-35nucleotides in length and at least one duplex region may be from 10-25nucleotides in length. The duplex may comprise two separate strands orit may comprise a single strand which comprises the first strand and thesecond strand.

The nucleic acid may: a) be blunt ended at both ends; b) have anoverhang at one end and a blunt end at the other; or c) have an overhangat both ends.

One or more nucleotides on the first and/or second strand may bemodified, to form modified nucleotides. One or more of the odd numberednucleotides of the first strand may be modified. One or more of the evennumbered nucleotides of the first strand may be modified by at least asecond modification, wherein the at least second modification isdifferent from the modification on the one or more odd numberednucleotides. At least one of the one or more modified even numberednucleotides may be adjacent to at least one of the one or more modifiedodd numbered nucleotides.

A plurality of odd numbered nucleotides in the first strand may bemodified in the nucleic acid disclosed herein. A plurality of evennumbered nucleotides in the first strand may be modified by a secondmodification. The first strand may comprise adjacent nucleotides thatare modified by a common modification. The first strand may alsocomprise adjacent nucleotides that are modified by a second differentmodification.

One or more of the odd numbered nucleotides of the second strand may bemodified by a modification that is different to the modification of theodd numbered nucleotides on the first strand and/or one or more of theeven numbered nucleotides of the second strand may be modified by thesame modification of the odd numbered nucleotides of the first strand.At least one of the one or more modified even numbered nucleotides ofthe second strand may be adjacent to the one or more modified oddnumbered nucleotides. A plurality of odd numbered nucleotides of thesecond strand may be modified by a common modification and/or aplurality of even numbered nucleotides may be modified by the samemodification that is present on the first strand odd numberednucleotides. A plurality of odd numbered nucleotides on the secondstrand may be modified by a second modification, wherein the secondmodification is different from the modification of the first strand oddnumbered nucleotides.

The second strand may comprise adjacent nucleotides that are modified bya common modification, which may be a second modification that isdifferent from the modification of the odd numbered nucleotides of thefirst strand.

In the nucleic acid disclosed herein, each of the odd numberednucleotides in the first strand and each of the even numberednucleotides in the second strand may be modified with a commonmodification and, each of the even numbered nucleotides may be modifiedin the first strand with a second modification and each of the oddnumbered nucleotides may be modified in the second strand with a seconddifferent modification.

The nucleic acid disclosed herein may have the modified nucleotides ofthe first strand shifted by at least one nucleotide relative to theunmodified or differently modified nucleotides of the second strand.

The modification and/or modifications may each and individually beselected from the group consisting of 3′-terminal deoxy-thymine,2′-O-methyl, a 2′-deoxy-modification, a 2′-amino-modification, a2′-alkyl-modification, a morpholino modification, a phosphoramidatemodification, 5′-phosphorothioate group modification, a 5′ phosphate or5′ phosphate mimic modification and a cholesteryl derivative or adodecanoic acid bisdecylamide group modification and/or the modifiednucleotide may be any one of a locked nucleotide, an abasic nucleotideor a non-natural base comprising nucleotide.

At least one modification may be 2′-O-methyl and/or at least onemodification may be 2′-F.

The disclosure further provides, as a second aspect, a nucleic acid forinhibiting expression of TMPRSS6, comprising at least one duplex regionthat comprises at least a portion of a first strand and at least aportion of a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of an RNA transcribed from theTMPRSS6 gene, wherein said first strand comprises a nucleotide sequenceselected from the following sequences:

SEQ ID Nos:

317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343,345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406,407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,436, 438, 440, or 442;

or selected from

SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 67,69, 71, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 85, 87, 88, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 270,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,299, 301, 309, 311, 312, 313, 314, or 315,

such as SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or215,

wherein the nucleotides of first strand are modified by firstmodification on the odd numbered nucleotides, and modified by a secondmodification on the even numbered nucleotides, and nucleotides of thesecond strand are modified by a third modification on the even numberednucleotides and modified by a fourth modification the odd numberednucleotides, wherein at least the first modification is different to thesecond modification and the third modification is different to thefourth modification.

The disclosure further provides, as a related second aspect, a nucleicacid for inhibiting expression of TMPRSS6, comprising at least oneduplex region that comprises at least a portion of a first strand and atleast a portion of a second strand that is at least partiallycomplementary to the first strand, wherein said first strand is at leastpartially complementary to at least a portion of an RNA transcribed fromthe TMPRSS6 gene, wherein said second strand may comprise a nucleotidesequence selected from the following sequences: SEQ ID no's 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 357,359. 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385,387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413,415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, or443,

or selected from SEQ ID no's 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 68, 70, 72, 75, 84, 86, 89, 100, 101, 102, 103, 104, 105,106, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,226, 228, 230, 232, 234, 236, 238, 240, 242, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 310, 312, 314, or 316,

such as a sequence selected from the following: SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, or 216,

wherein the nucleotides of first strand are modified by firstmodification on the odd numbered nucleotides, and modified by a secondmodification on the even numbered nucleotides, and nucleotides of thesecond strand are modified by a third modification on the even numberednucleotides and modified by a fourth modification the odd numberednucleotides, wherein at least the first modification is different to thesecond modification and the third modification is different to thefourth modification.

The third modification and the first modification may be the same and/orthe second modification and the fourth modification may be the same.

The first modification may be 2′OMe and the second modification may be2′F.

In the nucleic acid of any aspect, e.g. the second aspect, the firststrand may comprise the nucleotide sequence of SEQ ID NO:17 and thesecond strand may comprise the nucleotide sequence of SEQ ID NO:18. Thesequence and modifications may be as shown in the table below:

SEQ ID 5′aaccagaagaag 6273646282647284546 NO: 17 cagguga 3′ SEQ ID5′ucaccugcuucu 1727354715351718451 NO: 18 ucugguu 3′

wherein the specific modifications are depicted by the following numbers

1=2′F-dU,

2=2′F-dA,

3=2′F-dC,

4=2′F-dG,

5=2′-OMe-rU;

6=2′-OMe-rA;

7=2′-OMe-rC;

8=2′-OMe-rG.

As taught above, these are preferred sequences, along with the otherpreferred sequences.

A nucleic acid disclosed herein may comprise a phosphorothioate linkagebetween the terminal one, two or three 3′ nucleotides and/or one two orthree 5′ nucleotides of the first and/or the second strand. It maycomprise two phosphorothioate linkages between each of the threeterminal 3′ and between each of the three terminal 5′ nucleotides on thefirst strand, and two phosphorothioate linkages between the threeterminal nucleotides of the 3′ end of the second strand.

Such a nucleic acid may be conjugated to a ligand.

The disclosure further provides, as a third aspect, a nucleic acid forinhibiting expression of TMPRSS6, comprising at least one duplex regionthat comprises at least a portion of a first strand and at least aportion of a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of a RNA transcribed from theTMPRSS6 gene, wherein said first strand comprises a nucleotide sequenceselected from the following sequences,

SEQ ID Nos:

317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343,353 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404,406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432,434, 436, 438, 440, or 442;

or selected from

SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 67,69, 71, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 85, 87, 88, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 270,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,299, 301, 309, 311, 312, 313, 314, or 315,

such as SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or215,

and wherein the nucleic acid is conjugated to a ligand.

The disclosure further provides, as a further related aspect, a nucleicacid for inhibiting expression of TMPRSS6, comprising at least oneduplex region that comprises at least a portion of a first strand and atleast a portion of a second strand that is at least partiallycomplementary to the first strand, wherein said first strand is at leastpartially complementary to at least a portion of a RNA transcribed fromthe TMPRSS6 gene, wherein the second strand comprises a nucleotidesequence selected from the following sequences,

SEQ ID Nos 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,342, 344, 346, 357, 359. 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, or 443, or the second strand comprises a nucleotidesequence selected from the following sequences: SEQ ID no's 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 68, 70, 72, 75, 84, 86, 89,100, 101, 102, 103, 104, 105, 106, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 226, 228, 230, 232, 234, 236, 238, 240,242, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 310, 312, 314, or 316, such as a sequence selected from thefollowing: SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or216, and wherein the nucleic acid is conjugated to a ligand.

The ligand may comprise (i) one or more N-acetyl galactosamine (GalNAc)moieties and derivatives thereof, and (ii) a linker, wherein the linkerconjugates the GalNAc moieties to a sequence as defined in any precedingaspects. The linker may be a bivalent or trivalent or tetravalentbranched structure. The nucleotides may be modified as defined herein.

The ligand may comprise the formula I:

[S-X¹-P-X²]₃-A-linker-  (I)

wherein:

-   -   S represents a saccharide, wherein the saccharide is N-acetyl        galactosamine;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is alkylene or an alkylene ether of the formula        (—CH₂)_(n)—O—CH₂— where n=1-6;    -   A is a branching unit;    -   X³ represents a bridging unit;    -   wherein a nucleic acid disclosed herein is conjugated to X³ via        a    -   phosphate or modified phosphate (preferably a thiophosphate).

The present disclosure therefore additionally provides a conjugatednucleic acid having one of the following structures

wherein Z represents a nucleic acid as defined herein before.

Alternatively, a nucleic acid disclosed herein may be conjugated to aligand of the following structure

The invention also provides a composition comprising a nucleic acid asdefined herein in combination with one or more iron chelators and aformulation comprising:

-   -   i) a cationic lipid, or a pharmaceutically acceptable salt        thereof;    -   ii) a steroid;    -   iii) a phosphatidylethanolamine phospholipid;    -   iv) a PEGylated lipid.

The content of the cationic lipid component in the formulation may befrom about 55 mol % to about 65 mol % of the overall lipid content ofthe lipid formulation, preferably about 59 mol % of the overall lipidcontent of the lipid formulation.

The formulation may comprise a cationic lipid having the structure

a steroid having the structure

a phosphatidylethanolamine phospholipid having the structure

And a PEGylated lipid having the structure

Iron Chelators

An iron chelator is any molecule capable of binding to metal iron ionsin the body, and removing or decreasing excess iron from the body,thereby alleviating iron-mediated toxicity.

The one or more iron chelators may be selected from, for example,deferoxamine, deferiprone, deferasirox (Exjade) and deferairox (Jadenu),preferably deferiprone.

The invention also provides a composition comprising a nucleic acid or aconjugated nucleic acid of any aspect of the disclosure, in combinationwith one or more iron chelators, and a physiologically acceptableexcipient.

Also provided is a nucleic acid or a conjugated nucleic acid accordingto any aspect of the disclosure, in combination with one or more ironchelators, for use in the treatment of a disease or disorder and/or inthe manufacture of a medicament for treating a disease or disorder.

The invention provides a method of treating or preventing a disease ordisorder comprising administration of a composition comprising a nucleicacid or a conjugated nucleic acid according to any aspect of thedisclosure, in combination with one or more iron chelators, to anindividual in need of treatment.

The nucleic acid or conjugated nucleic acid may be administered to thesubject subcutaneously, intravenously or using any other applicationroutes such as oral, rectal or intraperitoneal.

The iron chelator may be administered to the subject subcutaneously,intravenously or using any other application routes such as oral, rectalor intraperitoneal.

The nucleic acid or conjugated nucleic acid may be administeredsimultaneously, separately or sequentially with one or more ironchelators.

It will therefore be appreciated that the nucleic acid or conjugatednucleic acid may be delivered in combination with an iron chelator,(such as a fixed dose composition) or the combination may be deliveredas separate pharmaceutical compositions.

The disease or disorder may be selected from the group comprisinghemochromatosis, porphyria cutanea tarda and blood disorders, such asβ-thalassemia or sickle cell disease, congenital dyserythropoieticanemia, marrow failure syndromes, myelodysplasia and transfusional ironoverload. The disorder may be associated with iron overload and thedisorder associated with iron overload may be Parkinson's Disease,Alzheimer's Disease or Friedreich's Ataxia. The disease or disorder mayalso be selected from infections and non-relapse related mortalityassociated with bone marrow transplantation. A preferred disease ordisorder to be treated with a nucleic acid as disclosed herein and oneor more iron chelators is hemochromatosis.

A method of making a nucleic acid or a conjugated nucleic acid accordingto the disclosure, in combination with one or more iron chelators, isalso included.

DETAILED DESCRIPTION OF INVENTION

The present invention relates to a nucleic acid which is double strandedand directed to an expressed RNA transcript of TMPRSS6 in combinationwith one or more iron chelators and compositions thereof. These nucleicacids can be used in the treatment of a variety of diseases anddisorders where reduced expression of TMPRSS6 gene product is desirable.The present invention provides a nucleic acid or conjugated nucleic acidaccording to any aspect disclosed herein in combination with one or moreiron chelators.

A first aspect disclosed herein relates to a nucleic acid for inhibitingexpression of TMPRSS6 in a cell, comprising at least one duplex regionthat comprises at least a portion of a first strand and at least aportion of a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of a RNA transcribed from theTMPRSS6 gene, wherein said first strand comprises a nucleotide sequenceselected from the following sequences:

SEQ ID Nos: 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339,341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374,376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402,404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430,432, 434, 436, 438, 440, or 442,

or selected from SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 67, 69, 71, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 85, 87,88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 225, 227, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,265, 267, 269, 270, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295, 297, 299, 301, 309, 311, 312, 313, 314, or 315,

such as SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or215.

By nucleic acid it is meant a nucleic acid comprising two strandscomprising nucleotides, that is able to interfere with gene expression.Inhibition may be complete or partial and results in down regulation ofgene expression in a targeted manner. The nucleic acid comprises twoseparate polynucleotide strands; the first strand, which may also be aguide strand; and a second strand, which may also be a passenger strand.The first strand and the second strand may be part of the samepolynucleotide molecule that is self complementary which ‘folds’ to forma double stranded molecule. The nucleic acid may be an siRNA molecule.

The nucleic acid may comprise ribonucleotides, modified ribonucleotides,deoxynucleotides, deoxyribonucleotides, or nucleotide analogues. Thenucleic acid may further comprise a double-stranded nucleic acid portionor duplex region formed by all or a portion of the first strand (alsoknown in the art as a guide strand) and all or a portion of the secondstrand (also known in the art as a passenger strand). The duplex regionis defined as beginning with the first base pair formed between thefirst strand and the second strand and ending with the last base pairformed between the first strand and the second strand, inclusive.

By duplex region it is meant the region in two complementary orsubstantially complementary oligonucleotides that form base pairs withone another, either by Watson-Crick base pairing or any other mannerthat allows for the formation of a duplex between oligonucleotidestrands that are complementary or substantially complementary. Forexample, an oligonucleotide strand having 21 nucleotide units can basepair with another oligonucleotide of 21 nucleotide units, yet only 19nucleotides on each strand are complementary or substantiallycomplementary, such that the “duplex region” consists of 19 base pairs.The remaining base pairs may exist as 5′ and 3′ overhangs, or as singlestranded regions. Further, within the duplex region, 100%complementarity is not required; substantial complementarity isallowable within a duplex region. Substantial complementarity refers tocomplementarity between the strands such that they are capable ofannealing under biological conditions. Techniques to empiricallydetermine if two strands are capable of annealing under biologicalconditions are well known in the art. Alternatively, two strands can besynthesised and added together under biological conditions to determineif they anneal to one another.

The portion of the first strand and second strand that form at least oneduplex region may be fully complementary and are at least partiallycomplementary to each other.

Depending on the length of a nucleic acid, a perfect match in terms ofbase complementarity between the first strand and second strand is notnecessarily required. However, the first and second strands must be ableto hybridise under physiological conditions.

The complementarity between the first strand and second strand in the atleast one duplex region may be perfect in that there are no nucleotidemismatches or additional/deleted nucleotides in either strand.Alternatively, the complementarity may not be perfect. Thecomplementarity may be at least 70%, 75%, 80%, 85%, 90% or 95%.

The first strand and the second strand may each comprise a region ofcomplementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the sequenceslisted in Table 1.

A related aspect of the disclosure relates to a nucleic acid forinhibiting expression of TMPRSS6 in a cell, comprising at least oneduplex region that comprises at least a portion of a first strand and atleast a portion of a second strand that is at least partiallycomplementary to the first strand, wherein said first strand is at leastpartially complementary to at least a portion of a RNA transcribed fromthe TMPRSS6 gene, wherein said second strand comprises a nucleotidesequence selected from the following sequences:

SEQ ID no's 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,342, 344, 346, 357, 359. 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, or 443,

or selected from

SEQ ID no's 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 68,70, 72, 75, 84, 86, 89, 100, 101, 102, 103, 104, 105, 106, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 226, 228, 230,232, 234, 236, 238, 240, 242, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,290, 292, 294, 296, 298, 300, 302, 310, 312, 314, or 316,

such as a sequence selected from the following: SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, or 216.

Suitably, in any aspect of the disclosure, the second and first standtogether are any of the complementary pairs of nucleic acids disclosedherein, such as those of SEQ ID 1 and 2, 3 and 4 etc.

The combination of nucleic acids consisting of SEQ ID 17 and 18 is apreferred combination, as is SEQ 199 with 200 and SEQ ID 207 with 208.

The nucleic acid involves the formation of a duplex region between allor a portion of the first strand and a portion of the target nucleicacid, TMPRSS6. The portion of the target nucleic acid that forms aduplex region with the first strand, defined as beginning with the firstbase pair formed between the first strand and the target sequence andending with the last base pair formed between the first strand and thetarget sequence, inclusive, is the target nucleic acid sequence orsimply, target sequence. The duplex region formed between the firststrand and the second strand need not be the same as the duplex regionformed between the first strand and the target sequence. That is, thesecond strand may have a sequence different from the target sequencehowever, the first strand must be able to form a duplex structure withboth the second strand and the target sequence.

The complementarity between the first strand and the target sequence maybe perfect (no nucleotide mismatches or additional/deleted nucleotidesin either nucleic acid).

The complementarity between the first strand and the target sequence maynot be perfect. The complementarity may be from about 70% to about 100%.More specifically, the complementarity may be at least 70%, 80%, 85%,90% or 95%, or an intermediate value.

The identity between the first strand and the complementary sequence ofthe target sequence may be from about 75% to about 100%. Morespecifically, the complementarity may be at least 75%, 80%, 85%, 90% or95%, or an intermediate value, provided a nucleic acid is capable ofreducing or inhibiting the expression of TMPRSS6.

A nucleic acid with less than 100% complementarity between the firststrand and the target sequence may be able to reduce the expression ofTMPRSS6 to the same level as a nucleic acid with perfect complementaritybetween the first strand and the target sequence. Alternatively, it maybe able to reduce expression of TMPRSS6 to a level that is 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the level of expressionachieved by the nucleic acid with perfect complementarity.

The nucleic acid may comprise a first strand and a second strand thatare each from 19-25 nucleotides in length. The first strand and thesecond strand may be of different lengths.

The nucleic acid may be 15-25 nucleotide pairs in length. The nucleicacid may be 17-23 nucleotide pairs in length. The nucleic acid may be17-25 nucleotide pairs in length. The nucleic acid may be 23-24nucleotide pairs in length. The nucleic acid may be 19-21 nucleotidepairs in length. The nucleic acid may be 21-23 nucleotide pairs inlength.

The nucleic acid may comprise a duplex region that consists of 19-25nucleotide base pairs. The duplex region may consist of 17, 18, 19, 20,21, 22, 23, 24 or 25 base pairs which may be contiguous.

The nucleic acid may comprise a first strand sequence of SEQ ID NO:17.The nucleic acid may comprise a second strand sequence of SEQ ID NO:18.The nucleic acid disclosed herein may comprise SEQ ID NO:17 and SEQ IDNO:18.

In a further aspect the nucleic acid as described herein may reduce theexpression of TMPRSS6 in a cell by at least 15% compared to the levelobserved in the absence of an inhibitor, which may be the nucleic acid.All preferred features of any of the previous aspects also apply to thisaspect. In particular, the expression of TMPRSS6 in a cell may bereduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or less, andintermediate values, than that observed in the absence of an inhibitor(which may be the nucleic acid).

The disclosure also relates to any first strand or any second strand ofnucleic acid as disclosed herein, which comprises no more than 2 basechanges when compared to the specific sequence ID provided. For example,one base may be changed within any sequence.

In one embodiment, the change may be made to the 5′ most nucleotide ofthe antisense (first) strand. In one embodiment, the change may be madeto the 3′ most nucleotide of the antisense (first) strand. In oneembodiment, the change may be made to the 5′ most nucleotide of thesense (second) strand. In one embodiment, the change may be made to the3′ most nucleotide of the sense (second) strand.

In one embodiment, the change is made to the 5′ most nucleotide of theantisense (first) strand. The base of the 5′ nucleotide may be changedto any other nucleotide. An A or a U at the 5′ end are preferred, and anA or a U are taught herein as the potential 5′ terminal base for all ofthe antisense (first strand) sequences disclosed herein

Overhangs

The nucleic acid may be blunt ended at both ends; have an overhang atone end and a blunt end at the other end; or have an overhang at bothends.

An “overhang” as used herein has its normal and customary meaning in theart, i.e. a single stranded portion of a nucleic acid that extendsbeyond the terminal nucleotide of a complementary strand in a doublestrand nucleic acid. The term “blunt end” includes double strandednucleic acid whereby both strands terminate at the same position,regardless of whether the terminal nucleotide(s) are base paired. Theterminal nucleotide of a first strand and a second strand at a blunt endmay be base paired. The terminal nucleotide of a first strand and asecond strand at a blunt end may not be paired. The terminal twonucleotides of a first strand and a second strand at a blunt end may bebase paired. The terminal two nucleotides of a first strand and a secondstrand at a blunt end may not be paired.

The nucleic acid may have an overhang at one end and a blunt end at theother. The nucleic acid may have an overhang at both ends. The nucleicacid may be blunt ended at both ends. The nucleic acid may be bluntended at the end with the 5′-end of the first strand and the 3′-end ofthe second strand or at the 3′-end of the first strand and the 5′-end ofthe second strand.

The nucleic acid may comprise an overhang at a 3′- or 5′-end. Thenucleic acid may have a 3′-overhang on the first strand. The nucleicacid may have a 3′-overhang on the second strand. The nucleic acid mayhave a 5′-overhang on the first strand. The nucleic acid may have a5′-overhang on the second strand. The nucleic acid may have an overhangat both the 5′-end and 3′-end of the first strand. The nucleic acid mayhave an overhang at both the 5′-end and 3′-end of the second strand. Thenucleic acid may have a 5′ overhang on the first strand and a 3′overhang on the second strand. The nucleic acid may have a 3′ overhangon the first strand and a 5′ overhang on the second strand. The nucleicacid may have a 3′ overhang on the first strand and a 3′ overhang on thesecond strand. The nucleic acid may have a 5′ overhang on the firststrand and a 5′ overhang on the second strand.

An overhang at the 3′-end or 5′ end of the second strand or the firststrand may be selected from consisting of 1, 2, 3, 4 and 5 nucleotidesin length. Optionally, an overhang may consist of 1 or 2 nucleotides,which may or may not be modified.

Modifications

Unmodified polynucleotides, particularly ribonucleotides, may be proneto degradation by cellular nucleases, and, as such,modifications/modified nucleotides may be included in the nucleic aciddisclosed herein.

One or more nucleotides on the second and/or first strand of the nucleicacid disclosed herein may be modified.

Modifications of the nucleic acid disclosed herein generally provide apowerful tool in overcoming potential limitations including, but notlimited to, in vitro and in vivo stability and bioavailability inherentto native RNA molecules. The nucleic acid according disclosed herein maybe modified by chemical modifications. Modified nucleic acid can alsominimise the possibility of inducing interferon activity in humans.Modification can further enhance the functional delivery of a nucleicacid to a target cell. The modified nucleic acid disclosed herein maycomprise one or more chemically modified ribonucleotides of either orboth of the first strand or the second strand. A ribonucleotide maycomprise a chemical modification of the base, sugar or phosphatemoieties. The ribonucleic acid may be modified by substitution orinsertion with analogues of nucleic acids or bases.

It will be appreciated that the disclosure of a modified nucleic acid,in particular such as an modified RNA, provides both a disclosure of the“primary” nucleic acid sequence, and also the modifications of thatsequence. A sequence listing thus provides both the information on theprimary nucleic acid sequence and also a modified sequence. For theavoidance of doubt, and the disclosure relates to both to unmodifiednucleic acid sequences, partially modified and any sequences for whichthe modifications have been fully defined.

One or more nucleotides of a nucleic acid disclosed herein may bemodified. The nucleic acid may comprise at least one modifiednucleotide. The modified nucleotide may be on the first strand. Themodified nucleotide may be in the second strand. The modified nucleotidemay be in the duplex region. The modified nucleotide may be outside theduplex region, i.e., in a single stranded region. The modifiednucleotide may be on the first strand and may be outside the duplexregion. The modified nucleotide may be on the second strand and may beoutside the duplex region. The 3′-terminal nucleotide of the firststrand may be a modified nucleotide. The 3′-terminal nucleotide of thesecond strand may be a modified nucleotide. The 5′-terminal nucleotideof the first strand may be a modified nucleotide. The 5′-terminalnucleotide of the second strand may be a modified nucleotide.

A nucleic acid disclosed herein may have 1 modified nucleotide or anucleic acid disclosed herein may have about 2-4 modified nucleotides,or a nucleic acid may have about 4-6 modified nucleotides, about 6-8modified nucleotides, about 8-10 modified nucleotides, about 10-12modified nucleotides, about 12-14 modified nucleotides, about 14-16modified nucleotides about 16-18 modified nucleotides, about 18-20modified nucleotides, about 20-22 modified nucleotides, about 22-24modified nucleotides, 24-26 modified nucleotides or about 26-28 modifiednucleotides. In each case the nucleic acid comprising said modifiednucleotides retains at least 50% of its activity as compared to the samenucleic acid but without said modified nucleotides. The nucleic acid mayretain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or anintermediate value of its activity as compared to the same nucleic acidbut without said modified nucleotides, or may have more than 100% of theactivity of the same nucleotide without said modified nucleotides.

The modified nucleotide may be a purine or a pyrimidine. At least halfof the purines may be modified. At least half of the pyrimidines may bemodified. All of the purines may be modified. All of the pyrimidines maybe modified. The modified nucleotides may be selected from the groupconsisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methylmodified nucleotide, a 2′ modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, a non-natural base comprisingnucleotide, a nucleotide comprising a 5′-phosphorothioate group, anucleotide comprising a 5′ phosphate or 5′ phosphate mimic and aterminal nucleotide linked to a cholesteryl derivative or a dodecanoicacid bisdecylamide group.

The nucleic acid may comprise a nucleotide comprising a modifiednucleotide, wherein the base is selected from 2-aminoadenosine,2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil,dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g.,5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

Nucleic acids discussed herein include unmodified RNA as well as RNAwhich has been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, for example as occur naturally in the human body. Modifiednucleotide as used herein refers to a nucleotide in which one or more ofthe components of the nucleic acid, namely sugars, bases, and phosphatemoieties, are different from that which occur in nature. While they arereferred to as modified nucleotides they will of course, because of themodification, include molecules which are not nucleotides, for example apolynucleotide molecules in which the ribophosphate backbone is replacedwith a non-ribophosphate construct that allows hybridisation betweenstrands i.e. the modified nucleotides mimic the ribophosphate backbone.

Many of the modifications described below that occur within a nucleicacid will be repeated within a polynucleotide molecule, such as amodification of a base, or a phosphate moiety, or the a non-linking 0 ofa phosphate moiety. In some cases the modification will occur at all ofthe possible positions/nucleotides in the polynucleotide but in manycases it will not. A modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal regions, such as at a position ona terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a nucleic acid disclosed herein or may only occur in asingle strand region of an nucleic acid disclosed herein. Aphosphorothioate modification at a non-linking 0 position may only occurat one or both termini, may only occur in a terminal region, e.g., at aposition on a terminal nucleotide or in the last 2, 3, 4 or 5nucleotides of a strand, or may occur in duplex and/or in single strandregions, particularly at termini. The 5′ end or 3′ ends may bephosphorylated.

Stability of a nucleic acid disclosed herein may be increased byincluding particular bases in overhangs, or by including modifiednucleotides, in single strand overhangs, e.g., in a 5′ or 3′ overhang,or in both. Purine nucleotides may be included in overhangs. All or someof the bases in a 3′ or 5′ overhang may be modified. Modifications caninclude the use of modifications at the 2′ OH group of the ribose sugar,the use of deoxyribonucleotides, instead of ribonucleotides, andmodifications in the phosphate group, such as phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the dsRNA agent disclosed herein may be phosphorylated. Insome embodiments, the overhang region contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

Nucleases can hydrolyze nucleic acid phosphodiester bonds. However,chemical modifications to nucleic acids can confer improved properties,and, can render oligoribonucleotides more stable to nucleases.

Modified nucleic acids, as used herein, can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygens(referred to as linking even if at the 5′ and 3′ terminus of the nucleicacid disclosed herein);

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar;

(iii) replacement of the phosphate moiety with “dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone;

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, e.g., a fluorescently labeled moiety, to either the 3′ or5′ end of RNA.

The terms replacement, modification, alteration, indicate a differencefrom a naturally occurring molecule.

Specific modifications are discussed in more detail below.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulphur. One, each or both non-linking oxygens in thephosphate group can be independently any one of S, Se, B, C, H, N, or OR(R is alkyl or aryl).

The phosphate linker can also be modified by replacement of a linkingoxygen with nitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at a terminal oxygen. Replacement of thenon-linking oxygens with nitrogen is possible.

A modified nucleotide can include modification of the sugar groups. The2′ hydroxyl group (OH) can be modified or replaced with a number ofdifferent “oxy” or “deoxy” substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “locked” nucleicacids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylenebridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino)and aminoalkoxy, O(CH₂)nAMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino).

“Deoxy” modifications include hydrogen halo; amino (e.g., NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid);NH(CH₂CH₂NH)nCH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl,cycloalkyl, aryl, alkenyl and alkynyl, which may be optionallysubstituted with e.g., an amino functionality. Other substitutents ofcertain embodiments include 2′-methoxyethyl, 2′-OCH₃, 2′-O-allyl,2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified nucleotides may contain a sugar suchas arabinose.

Modified nucleotides can also include “abasic” sugars, which lack anucleobase at C—I′. These abasic sugars can further containmodifications at one or more of the constituent sugar atoms.

The 2′ modifications may be used in combination with one or morephosphate linker modifications (e.g., phosphorothioate).

The phosphate group can be replaced by non-phosphorus containingconnectors.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.In certain embodiments, replacements may include themethylenecarbonylamino and methylenemethylimino groups.

The phosphate linker and ribose sugar may be replaced by nucleaseresistant nucleotides. Examples include the morpholino, cyclobutyl,pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. Incertain embodiments, PNA surrogates may be used.

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end or the 5′ end or both ends of themolecule. They can include modification or replacement of an entireterminal phosphate or of one or more of the atoms of the phosphategroup. For example, the 3′ and 5′ ends of an oligonucleotide can beconjugated to other functional molecular entities such as labelingmoieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 orCy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron orester). The functional molecular entities can be attached to the sugarthrough a phosphate group and/or a linker. The terminal atom of thelinker can connect to or replace the linking atom of the phosphate groupor the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, thelinker can connect to or replace the terminal atom of a nucleotidesurrogate (e.g., PNAs). These spacers or linkers can include e.g.,—(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—,O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6), abasic sugars, amide, carboxy,amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide,or morpholino, or biotin and fluorescein reagents. The 3′ end can be an—OH group.

Other examples of terminal modifications include dyes, intercalatingagents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O³-(oleoyl)lithocholic acid,O³-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g., biotin), transport/absorption facilitators (e.g.,aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,imidazole, bisimidazole, histamine, imidazole clusters,acridine-imidazole conjugates, Eu³⁺ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingto modulate activity or to modulate resistance to degradation. Terminalmodifications useful for modulating activity include modification of the5′ end with phosphate or phosphate analogs. Nucleic acids disclosedherein, on the first or second strand, may be 5′ phosphorylated orinclude a phosphoryl analog at the 5′ prime terminus. 5′-phosphatemodifications include those which are compatible with RISC mediated genesilencing. Suitable modifications include: 5′-monophosphate((HO)₂(O)P—O-5′); 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap(7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate(phosphorothioate; (HO)₂(S)P—O-5′); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.,5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g., RP(OH)(O)—O-5′-, (OH)₂(O)P-5′—CH₂—), 5′vinylphosphonate,5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH₂—),ethoxymethyl, etc., e.g., RP(OH)(O)—O-5′-).

The nucleic acid of the present disclosure may include one or morephosphorothioate modifications on one or more of the terminal ends ofthe first and/or the second strand. Optionally, each or either end ofthe first strand may comprise one or two or three phosphorothioatemodified nucleotides. Optionally, each or either end of the secondstrand may comprise one or two or three phosphorothioate modifiednucleotides. Optionally, both ends of the first strand and the 5′ end ofthe second strand may comprise two phosphorothioate modifiednucleotides. By phosphorothioate modified nucleotide it is meant thatthe linkage between the nucleotide and the adjacent nucleotide comprisesa phosphorothioate group instead of a standard phosphate group.

Terminal modifications can also be useful for monitoring distribution,and in such cases the groups to be added may include fluorophores, e.g.,fluorescein or an Alexa dye. Terminal modifications can also be usefulfor enhancing uptake, useful modifications for this include cholesterol.Terminal modifications can also be useful for cross-linking an RNA agentto another moiety.

Nucleotides

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the above basesand “universal bases” can be employed. Examples include 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and 0-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N<4>-acetyl cytosine,2-thiocytosine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases.

As used herein, the terms “non-pairing nucleotide analogue” means anucleotide analogue which includes a non-base pairing moiety includingbut not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analog is a ribonucleotide.In other embodiments it is a deoxyribonucleotide.

As used herein, the term, “terminal functional group” includes withoutlimitation a halogen, alcohol, amine, carboxylic, ester, amide,aldehyde, ketone, ether groups.

Certain moieties may be linked to the 5′ terminus of the first strand orthe second strand and includes abasic ribose moiety, abasic deoxyribosemoiety, modifications abasic ribose and abasic deoxyribose moietiesincluding 2′ O alkyl modifications; inverted abasic ribose and abasicdeoxyribose moieties and modifications thereof, C6-imino-Pi; a mirrornucleotide including L-DNA and L-RNA; 5′OMe nucleotide; and nucleotideanalogs including 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non bridging methylphosphonate and 5′-mercaptomoieties.

The nucleic acids disclosed herein may be included one or more invertednucleotides, for example inverted thymidine or inverted adenine (forexample see Takei, et al., 2002. JBC 277 (26):23800-06).

As used herein, the term “inhibit”, “down-regulate”, or “reduce” withrespect to gene expression means the expression of the gene, or level ofRNA molecules or equivalent RNA molecules encoding one or more proteinsor protein subunits (e.g., mRNA), or activity of one or more proteins,protein subunits or peptides, is reduced below that observed in theabsence of a nucleic acid disclosed herein or in reference to an siRNAmolecule with no known homology to human transcripts (herein termednon-silencing control). Such control may be conjugated and modified inan analogous manner to the molecule disclosed herein and delivered intothe target cell by the same route; for example the expression may bereduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or anintermediate value, in the absence of the nucleic acid or conjugatednucleic acid disclosed herein, or in the presence of a non-silencingcontrol.

The nucleic acid of the present disclosure may comprise an abasicnucleotide. The term “abasic” as used herein, refers to moieties lackinga base or having other chemical groups in place of a base at the 1′position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribosederivative.

The nucleic acid may comprise one or more nucleotides on the secondand/or first strands that are modified. Alternating nucleotides may bemodified, to form modified nucleotides.

Alternating as described herein means to occur one after another in aregular way. In other words, alternating means to occur in turnrepeatedly. For example if one nucleotide is modified, the nextcontiguous nucleotide is not modified and the following contiguousnucleotide is modified and so on. One nucleotide may be modified with afirst modification, the next contiguous nucleotide may be modified witha second modification and the following contiguous nucleotide ismodified with the first modification and so on, where the first andsecond modifications are different.

One or more of the odd numbered nucleotides of the first strand of thenucleic acid disclosed herein may be modified wherein the first strandis numbered 5′ to 3′. The term “odd numbered” as described herein meansa number not divisible by two. Examples of odd numbers are 1, 3, 5, 7,9, 11 and so on. One or more of the even numbered nucleotides of thefirst strand of the nucleic acid disclosed herein may be modified,wherein the first strand is numbered 5′ to 3′. The term “even numbered”as described herein means a number which is evenly divisible by two.Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on. One ormore of the odd numbered nucleotides of the second strand of the nucleicacid disclosed herein may be modified wherein the second strand isnumbered 3′ to 5′. One or more of the even numbered nucleotides of thesecond strand of the nucleic acid disclosed herein may be modified,wherein the second strand is numbered 3′ to 5′.

One or more nucleotides on the first and/or second strand may bemodified, to form modified nucleotides. One or more of the odd numberednucleotides of the first strand may be modified. One or more of the evennumbered nucleotides of the first strand may be modified by at least asecond modification, wherein the at least second modification isdifferent from the modification on the one or more add nucleotides. Atleast one of the one or more modified even numbered nucleotides may beadjacent to at least one of the one or more modified odd numberednucleotides.

A plurality of odd numbered nucleotides in the first strand may bemodified in the nucleic acid disclosed herein. A plurality of evennumbered nucleotides in the first strand may be modified by a secondmodification. The first strand may comprise adjacent nucleotides thatare modified by a common modification. The first strand may alsocomprise adjacent nucleotides that are modified by a second differentmodification.

One or more of the odd numbered nucleotides of the second strand may bemodified by a modification that is different to the modification of theodd numbered nucleotides on the first strand and/or one or more of theeven numbered nucleotides of the second strand may be by the samemodification of the odd numbered nucleotides of the first strand. Atleast one of the one or more modified even numbered nucleotides of thesecond strand may be adjacent to the one or more modified odd numberednucleotides. A plurality of odd numbered nucleotides of the secondstrand may be modified by a common modification and/or a plurality ofeven numbered nucleotides may be modified by the same modification thatis present on the first stand odd numbered nucleotides. A plurality ofodd numbered nucleotides on the second strand may be modified by asecond modification, wherein the second modification is different fromthe modification of the first strand odd numbered nucleotides.

The second strand may comprise adjacent nucleotides that are modified bya common modification, which may be a second modification that isdifferent from the modification of the odd numbered nucleotides of thefirst strand.

In the nucleic acid disclosed herein, each of the odd numberednucleotides in the first strand and each of the even numberednucleotides in the second strand may be modified with a commonmodification and, each of the even numbered nucleotides may be modifiedin the first strand with a second modification and each of the oddnumbered nucleotides may be modified in the second strand with thesecond modification.

The nucleic acid disclosed herein may have the modified nucleotides ofthe first strand shifted by at least one nucleotide relative to theunmodified or differently modified nucleotides of the second strand.

One or more or each of the odd numbered nucleotides may be modified inthe first strand and one or more or each of the even numberednucleotides may be modified in the second strand. One or more or each ofthe alternating nucleotides on either or both strands may be modified bya second modification. One or more or each of the even numberednucleotides may be modified in the first strand and one or more or eachof the even numbered nucleotides may be modified in the second strand.One or more or each of the alternating nucleotides on either or bothstrands may be modified by a second modification. One or more or each ofthe odd numbered nucleotides may be modified in the first strand and oneor more of the odd numbered nucleotides may be modified in the secondstrand by a common modification. One or more or each of the alternatingnucleotides on either or both strands may be modified by a secondmodification. One or more or each of the even numbered nucleotides maybe modified in the first strand and one or more or each of the oddnumbered nucleotides may be modified in the second strand by a commonmodification. One or more or each of the alternating nucleotides oneither or both strands may be modified by a second modification.

The nucleic acid disclosed herein may comprise single or double strandedconstructs that comprise at least two regions of alternatingmodifications in one or both of the strands. These alternating regionscan comprise up to about 12 nucleotides but preferably comprise fromabout 3 to about 10 nucleotides. The regions of alternating nucleotidesmay be located at the termini of one or both strands of the nucleic aciddisclosed herein. The nucleic acid may comprise from 4 to about 10nucleotides of alternating nucleotides at each termini (3′ and 5′) andthese regions may be separated by from about 5 to about 12 contiguousunmodified or differently or commonly modified nucleotides.

The odd numbered nucleotides of the first strand may be modified and theeven numbered nucleotides may be modified with a second modification.The second strand may comprise adjacent nucleotides that are modifiedwith a common modification, which may be the same as the modification ofthe odd numbered nucleotides of the first strand. One or morenucleotides of second strand may also be modified with the secondmodification. One or more nucleotides with the second modification maybe adjacent to each other and to nucleotides having a modification thatis the same as the modification of the odd numbered nucleotides of thefirst strand. The first strand may also comprise phosphorothioatelinkages between the two nucleotides at the 3′ end and at the 5′ end.The second strand may comprise a phosphorothioate linkage between thetwo nucleotides at 5′ end. The second strand may also be conjugated to aligand at the 5′ end.

The nucleic acid disclosed herein may comprise a first strand comprisingadjacent nucleotides that are modified with a common modification. Oneor more of such nucleotides may be adjacent to one or more nucleotideswhich may be modified with a second modification. One or morenucleotides with the second modification may be adjacent. The secondstrand may comprise adjacent nucleotides that are modified with a commonmodification, which may be the same as one of the modifications of oneor more nucleotides of the first strand. One or more nucleotides ofsecond strand may also be modified with the second modification. One ormore nucleotides with the second modification may be adjacent. The firststrand may also comprise phosphorothioate linkages between the twonucleotides at the 5′ end and at the 3′ end. The second strand maycomprise a phosphorothioate linkage between the two nucleotides at 3′end. The second strand may also be conjugated to a ligand at the 5′ end.

The nucleotides numbered from 5′ to 3′ on the first strand and 3′ and 5′on the second strand, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25may be modified by a modification on the first strand. The nucleotidesnumbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modifiedby a second modification on the first strand. The nucleotides numbered1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by amodification on the second strand. The nucleotides numbered 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a secondmodification on the second strand. Nucleotides are numbered for the sakeof the nucleic acid of the present disclosure from 5′ to 3′ on the firststrand and 3′ and 5′ on the second strand

The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24may be modified by a modification on the first strand. The nucleotidesnumbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by asecond modification on the first strand. The nucleotides numbered 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification onthe second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22 and 24 may be modified by a second modification on the secondstrand.

Clearly, if the first and/or the second strand are shorter than 25nucleotides in length, such as 19 nucleotides in length, there are nonucleotides numbered 20, 21, 22, 23, 24 and 25 to be modified. Theskilled person understands the description above to apply to shorterstrands, accordingly.

One or more modified nucleotides on the first strand may be paired withmodified nucleotides on the second strand having a common modification.One or more modified nucleotides on the first strand may be paired withmodified nucleotides on the second strand having a differentmodification. One or more modified nucleotides on the first strand maybe paired with unmodified nucleotides on the second strand. One or moremodified nucleotides on the second strand may be paired with unmodifiednucleotides on the first strand. In other words, the alternatingnucleotides can be aligned on the two strands such as, for example, allthe modifications in the alternating regions of the second strand arepaired with identical modifications in the first strand or alternativelythe modifications can be offset by one nucleotide with the commonmodifications in the alternating regions of one strand pairing withdissimilar modifications (i.e. a second or further modification) in theother strand. Another option is to have dissimilar modifications in eachof the strands.

The modifications on the first strand may be shifted by one nucleotiderelative to the modified nucleotides on the second strand, such thatcommon modified nucleotides are not paired with each other.

The modification and/or modifications may each and individually beselected from the group consisting of 3′-terminal deoxy-thymine,2′-O-methyl, a 2′-deoxy-modification, a 2′-amino-modification, a2′-alkyl-modification, a morpholino modification, a phosphoramidatemodification, 5′-phosphorothioate group modification, a 5′ phosphate or5′ phosphate mimic modification and a cholesteryl derivative or adodecanoic acid bisdecylamide group modification and/or the modifiednucleotide may be any one of a locked nucleotide, an abasic nucleotideor a non-natural base comprising nucleotide.

At least one modification may be 2′-O-methyl and/or at least onemodification may be 2′-F.

Further modifications as described herein may be present on the firstand/or second strand.

Throughout the description of the invention and disclosure, “same orcommon modification” means the same modification to any nucleotide, bethat A, G, C or U modified with a group such as such as a methyl groupor a fluoro group. Is it not taken to mean the same addition on the samenucleotide. For example, 2′F-dU, 2′F-dA, 2′F-dC, 2′F-dG are allconsidered to be the same or common modification, as are 2′-OMe-rU,2′-OMe-rA; 2′-OMe-rC; 2′-OMe-rG. A 2′F modification is a differentmodification to a 2′OMe modification.

Some representative modified nucleic acid sequences of the presentdisclosure are shown in the examples. These examples are meant to berepresentative and not limiting.

Preferably, the nucleic acid may comprise a modification and the secondor further modification which are each and individually selected fromthe group comprising 2′-O-methyl modification and 2′-F modification. Thenucleic acid may comprise a modification that is 2′-O-methyl (2′OMe)that may be a first modification, and a second modification that is2′-F. The nucleic acid disclosed herein may also include aphosphorothioate modification and/or a deoxy modification which may bepresent in or between the terminal 1, 2 or 3 nucleotides of each or anyend of each or both strands.

The disclosure provides as a further aspect, a nucleic acid forinhibiting expression of TMPRSS6, comprising a nucleotide sequence ofSEQ ID 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404,406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432,434, 436, 438, 440, or 442, or SEQ ID 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 67, 69, 71, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83,85, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, 265, 267, 269, 270, 271, 273, 275, 277, 279, 281, 283, 285, 287,289, 291, 293, 295, 297, 299, 301, 309, 311, 312, 313, 314, or 315, suchas SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215,

wherein the nucleotides of first strand are modified by a firstmodification on the odd numbered nucleotides, and modified by a secondmodification on the even numbered nucleotides, and nucleotides of thesecond strand are modified by a third modification on the even numberednucleotides and modified by a fourth modification the odd numberednucleotides, wherein at least the first modification is different to thesecond modification and the third modification is different to thefourth modification. The third and first modifications may be the sameor different, the second and fourth modifications may be the same ordifferent. The first and second modifications may be different to eachother and the third and fourth modifications may be different to eachother.

In a further aspect is provided a nucleic acid for inhibiting expressionof TMPRSS6, wherein the second strand comprises a nucleotide sequence ofSEQ ID NO 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,342, 344, 346, 357, 359. 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, or 443, or a nucleotide sequence of SEQ ID no 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 68, 70, 72, 75, 84,86, 89, 100, 101, 102, 103, 104, 105, 106, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 226, 228, 230, 232, 234, 236, 238,240, 242, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 310, 312, 314, or 316, such as a nucleotide sequence ofSEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216

wherein the nucleotides of first strand are modified by a firstmodification on the odd numbered nucleotides, and modified by a secondmodification on the even numbered nucleotides, and nucleotides of thesecond strand are modified by a third modification on the even numberednucleotides and modified by a fourth modification the odd numberednucleotides, wherein at least the first modification is different to thesecond modification and the third modification is different to thefourth modification. The third and first modifications may be the sameor different, the second and fourth modifications may be the same ordifferent. The first and second modifications may be different to eachother and the third and fourth modifications may be different to eachother. Suitably the nucleotide sequence of the second strand iscomplementary to the first strand. The nucleotides of first strand maybe modified by first modification on the odd numbered nucleotides, andmodified with a second modification on the even numbered nucleotides,and the second strand may be modified on the odd numbered nucleotideswith the second modification and modified with the first modificationthe even numbered nucleotides. The first modification may be 2′OMe andthe second modification may be 2′ F. The first strand may comprise thenucleotide sequence of SEQ ID NO: 17 and/or the second strand maycomprise the nucleotide sequence of SEQ ID NO: 18. The modifications maybe those as set out in table 1.

In one aspect all the even-numbered nucleotides of the first strand ofthe nucleic acid are modified by a first modification, all theodd-numbered nucleotides of the first strand are modified by a secondmodification, all the nucleotides of the second strand in positionscorresponding to nucleotides 11-13 of the first strand are modified by afourth modification, all the nucleotides of the second strand other thanthe nucleotides corresponding to nucleotides 11-13 of the first strandare modified by a third modification, wherein the first and fourthmodification are preferably 2′-F and the second and third modificationare preferably 2′-OMe.

The nucleic acid disclosed herein may be conjugated to a ligand.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. The endosomolytic component may contain a chemical groupwhich undergoes a change in charge or protonation in response to achange in pH. The endosomolytic component may be linear or branched.

Ligands can include therapeutic modifiers, e.g., for enhancing uptake;diagnostic compounds or reporter groups e.g., for monitoringdistribution; cross-linking agents; and nuclease-resistance conferringmoieties. General examples include lipids, steroids, vitamins, sugars,proteins, peptides, polyamines, and peptide mimics. Ligands can includea naturally occurring substance, such as a protein, carbohydrate, orlipid. The ligand may be a recombinant or synthetic molecule.

Ligands can also include targeting groups, e.g. a cell or tissuetargeting agent. The targeting ligand may be a lectin, glycoprotein,lipid or protein.

Other examples of ligands include dyes, intercalating agents,cross-linkers, porphyrins, polycyclic aromatic hydrocarbons, artificialendonucleases or a chelator, lipophilic molecules, alkylating agents,phosphate, amino, mercapto, PEG, MPEG, alkyl, substituted alkyl,radiolabelled markers, enzymes, haptens, transport/absorptionfacilitators, synthetic ribonucelases, or imidazole clusters.

Ligands can be proteins, e.g. glycoproteins or peptides. Ligands mayalso be hormones or hormone receptors. They may also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,or cofactors.

The ligand may be a substance such as a drug which can increase theuptake of the nucleic acid into a cell, for example, by disrupting thecell's cytoskeleton.

The ligand may increase uptake of the nucleic acid into the cell byactivating an inflammatory response. Such ligands include tumournecrosis factor alpha (TNF-alpha), interleukin-1 beta, or gammainterferon.

The ligand may be a lipid or lipid-based molecule. The lipid orlipid-based molecule preferably binds a serum protein. Preferably, thelipid-based ligand binds human serum albumin (HSA). A lipid orlipid-based molecule can increase resistance to degradation of theconjugate, increase targeting or transport into target cell, and/or canadjust binding to a serum protein. A lipid-based ligand can be used tomodulate binding of the conjugate to a target tissue.

The ligand may be a steroid. Preferably, the ligand is cholesterol or acholesterol derivative.

The ligand may be a moiety e.g. a vitamin, which is taken up by a targetcell. Exemplary vitamins include vitamin A, E, K, and the B vitamins.Vitamins may be taken up by a proliferating cell, which may be usefulfor delivering the nucleic acid to cells such as malignant ornon-malignant tumour cells.

The ligand may be a cell-permeation agent, such as a helicalcell-permeation agent. Preferably such an agent is amphipathic.

The ligand may be a peptide or peptidomimetic. A peptidomimetic is amolecule capable of folding into a defined three-dimensional structuresimilar to a natural peptide. The peptide or peptidomimetic ligand mayinclude naturally occurring or modified peptides, or both. A peptide orpeptidomimetic can be a cell permeation peptide, cationic peptide,amphipathic peptide, or hydrophobic peptide. The peptide moiety can be adendrimer peptide, constrained peptide, or crosslinked peptide. Thepeptide moiety can include a hydrophobic membrane translocationsequence. The peptide moiety can be a peptide capable of carrying largepolar molecules such as peptides, oligonucleotides, and proteins acrosscell membranes, e.g. sequences from the HIV Tat protein (GRKKRRQRRRPPQ)and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK). Preferablythe peptide or peptidomimetic is a cell targeting peptide, e.g.arginine-glycine-aspartic acid (RGD)-peptide.

The ligand may be a cell permeation peptide that is capable ofpermeating, for example, a microbial cell or a mammalian cell.

The ligand may be a pharmacokinetic modulator. The pharmacokineticmodulator may be lipophiles, bile acids, steroids, phospholipidanalogues, peptides, protein binding agents, PEG, vitamins, etc.

When two or more ligands are present, the ligands can all have the sameproperties, all have different properties, or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the nucleic acid at the 3′-end, 5′-end, and/orat an internal position. Preferably the ligand is coupled to the nucleicacid via an intervening tether or linker.

In some embodiments the nucleic acid is a double-stranded nucleic acid.In a double-stranded nucleic acid the ligand may be attached to one orboth strands. In some embodiments, a double-stranded nucleic acidcontains a ligand conjugated to the sense strand. In other embodiments,a double-stranded nucleic acid contains a ligand conjugated to theantisense strand.

Ligands can be conjugated to nucleobases, sugar moieties, orinternucleosidic linkages of nucleic acid molecules. Conjugation topurine nucleobases or derivatives thereof can occur at any positionincluding endocyclic and exocyclic atoms. Conjugation to pyrimidinenucleotides or derivatives thereof can also occur at any position.Conjugation to sugar moieties of nucleosides can occur at any carbonatom. Conjugation to internucleosidic linkages may occur at thephosphorus atom of a phosphorus-containing linkage or at an oxygen,nitrogen, or sulphur atom bonded to the phosphorus atom. For amine- oramide-containing internucleosidic linkages, conjugation may occur at thenitrogen atom of the amine or amide or to an adjacent carbon atom.

The ligand is typically a carbohydrate, e.g. a monosaccharide,disaccharide, trisaccharide, tetrasaccharide or polysaccharide. Theligand may be conjugated to the nucleic acid by a linker. The linker maybe a monovalent, bivalent, or trivalent branched linker.

Efficient delivery of oligonucleotides, in particular double strandednucleic acids disclosed herein, to cells in vivo is important andrequires specific targeting and substantial protection from theextracellular environment, particularly serum proteins. One method ofachieving specific targeting is to conjugate a ligand to the nucleicacid. The ligand helps in targeting the nucleic acid to the requiredtarget site. There is a need to conjugate appropriate ligands for thedesired receptor molecules in order for the conjugated molecules to betaken up by the target cells by mechanisms such as differentreceptor-mediated endocytosis pathways or functionally analogousprocesses. The targeting moiety or ligand can be any moiety or ligandthat is capable of targeting a specific receptor.

For example, the Asialoglycoprotein receptor (ASGP-R) is a high capacityreceptor, which is highly abundant on hepatocytes. One of the firstdisclosures of triantennary cluster glycosides was in U.S. Pat. No.5,885,968. Conjugates having three GalNAc ligands and comprisingphosphate groups are known and are described in Dubber et al. (2003,Bioconjug. Chem. 2003 January-February; 14(1):239-46.). The ASGP-R showsa 50-fold higher affinity for N-Acetyl-D-Galactosylamine (GalNAc) thanD-Gal.

Hepatocytes expressing the lectin (asialoglycoprotein receptor; ASGPR),which recognizes specifically terminal β-galactosyl subunits ofglycosylated proteins or other oligosaccharides (Weigel, P. H. et. al.,Biochim. Biophys. Acta. 2002 Sep. 19; 1572(2-3):341-63), can be used fortargeting a drug to the liver by covalent coupling of galactose orgalactoseamine to the drug substance (Ishibashi, S.; et. al., J Biol.Chem. 1994 Nov. 11; 269(45):27803-6)). Furthermore the binding affinitycan be significantly increased by the multi-valency effect, which isachieved by the repetition of the targeting unit (E. A. L. Biessen et.al., 1995).

The ASGPR is a mediator for an active endosomal transport of terminalβ-galactosyl containing glycoproteins, thus ASGPR is highly suitable fortargeted delivery of drug candidates like nucleic acid, which have to bedelivered into a cell (Akinc et al.).

The ligand may be attached to the nucleic acid disclosed herein via alinker, which may be a bivalent or trivalent or tetramer branchedlinker.

The saccharide, which can also be referred to as the ligand, may beselected to have an affinity for at least one type of receptor on atarget cell. In particular, the receptor is on the surface of amammalian liver cell, for example, the hepatic asialoglycoproteinreceptor (ASGP-R).

The saccharide may be selected from N-acetyl galactosamine, mannose,galactose, glucose, glucosamine and fucose. The saccharide may beN-acetyl galactosamine (GalNAc).

A ligand for use in the present disclosure may therefore comprise (i)one or more N-acetyl galactosamine (GalNAc) moieties and derivativesthereof, and (ii) a linker, wherein the linker conjugates the GalNAcmoieties to a sequence as defined in any preceding aspects. The linkermay be a bivalent or trivalent or tetravalent branched structure. Thenucleotides may be modified as defined herein.

“GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonlyreferred to in the literature as N-acetyl galactosamine. Reference to“GalNAc” or “N-acetyl galactosamine” includes both the β-form:2-(Acetylamino)-2-deoxy-β-D-galactopyranose and the α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose. Both the β-form:2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably.Preferably, the compounds disclosed herein comprise the β-form,2-(Acetylamino)-2-deoxy-β-D-galactopyranose.

The ligand may comprise GalNAc.

The ligand may comprise a compound of formula I:

[S-X¹-P-X²]₃-A-linker-  (I)

wherein:

-   -   S represents a saccharide, wherein the saccharide is N-acetyl        galactosamine;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is alkylene or an alkylene ether of the formula        (—CH₂)_(n)—O—CH₂— where n=1-6;    -   A is a branching unit;    -   X³ represents a bridging unit;    -   wherein a nucleic acid disclosed herein is conjugated to X³ via        a phosphate or modified phosphate (preferably a thiophosphate).

In formula I, branching unit “A” branches into three in order toaccommodate the three saccharide moieties. The branching unit iscovalently attached to the remaining tethered portions of the ligand andthe nucleic acid. The branching unit may comprise a branched aliphaticgroup comprising groups selected from alkyl, amide, disulphide,polyethylene glycol, ether, thioether and hydroxyamino groups. Thebranching unit may comprise groups selected from alkyl and ether groups.

The branching unit A may have a structure selected from:

wherein each A₁ independently represents O, S, C═O or NH; and

each n independently represents an integer from 1 to 20.

The branching unit may have a structure selected from:

wherein each A₁ independently represents O, S, C═O or NH; and

each n independently represents an integer from 1 to 20.

The branching unit may have a structure selected from:

wherein A₁ is O, S, C═O or NH; and

each n independently represents an integer from 1 to 20.

The branching unit may have the structure:

The branching unit may have the structure:

The branching unit may have the structure:

Optionally, the branching unit consists of only a carbon atom.

The “X³” portion of the compounds of formula I is a bridging unit. Thebridging unit is linear and is covalently bound to the branching unitand the nucleic acid.

X³ may be selected from —C₁-C₂₀ alkylene-, —C₂-C₂₀ alkenylene-, analkylene ether of formula —(C₁-C₂₀ alkylene)-O—(C₁-C₂₀ alkylene)-,—C(O)—C₁-C₂₀ alkylene-, —C₀-C₄ alkylene(Cy)C₀-C₄ alkylene- wherein Cyrepresents a substituted or unsubstituted 5 or 6 membered cycloalkylene,arylene, heterocyclylene or heteroarylene ring, —C₁-C₄alkylene-NHC(O)—C₁-C₄ alkylene-, —C₁-C₄ alkylene-C(O)NH—C₁-C₄ alkylene-,—C₁-C₄ alkylene-SC(O)—C₁-C₄ alkylene-, —C₁-C₄ alkylene-C(O)S—C₁-C₄alkylene-, —C₁-C₄ alkylene-OC(O)—C₁-C₄ alkylene-, —C₁-C₄alkylene-C(O)O—C₁-C₄ alkylene-, and —C₁-C₆ alkylene-S—S—C₁-C₆ alkylene-.

X³ may be an alkylene ether of formula —(C₁-C₂₀ alkylene)-O—(C₁-C₂₀alkylene)-. X³ may be an alkylene ether of formula —(C₁-C₂₀alkylene)-O—(C₄-C₂₀ alkylene)-, wherein said (C₄-C₂₀ alkylene) is linkedto Z. X³ may be selected from the group consisting of —CH₂—O—C₃H₆—,—CH₂—O—C₄H₆—, —CH₂—O—C₆H₁₂— and —CH₂—O—CH₁₆—, especially —CH₂—O—C₄H₈—,—CH₂—O—C₆H₁₂— and —CH₂—O—CH₁₆—, wherein in each case the —CH₂— group islinked to A.

The ligand may comprise a compound of formula (II):

[S-X¹-P-X²]₃-A-X³-  (II)

wherein:

-   -   S represents a saccharide;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is C₁-C₈ alkylene;    -   A is a branching unit selected from:

-   -   X³ is a bridging unit;    -   wherein a nucleic acid disclosed herein is conjugated to X³ via        a phosphate or modified phosphate (preferably a thiophosphate).

Branching unit A may have the structure:

Branching unit A may have the structure:

wherein X³ is attached to the nitrogen atom.

X³ may be C₁-C₂₀ alkylene. Preferably, X³ is selected from the groupconsisting of —C₃H₆—, —C₄H₈—, —C₆H₁₂— and —C₈H₁₆—, especially —C₄H₈—,—C₆H₁₂— and —C₈H₁₆—.

The ligand may comprise a compound of formula (III):

[S-X¹-P-X²]₃-A-X³-  (III)

wherein:

-   -   S represents a saccharide;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is an alkylene ether of formula —C₃H₆—O—CH₂—;    -   A is a branching unit;    -   X³ is an alkylene ether of formula selected from the group        consisting of —CH₂—O—CH₂—, —CH₂—O—C₂H₄—, —CH₂—O—C₃H₆—,        —CH₂—O—C₄H₈—, —CH₂—O—C₅H₁₀—, —CH₂—O—C₆H₁₂—, —CH₂—O—C₇H₁₄—, and        —CH₂—O—C₈H₁₆—, wherein in each case the —CH₂— group is linked to        A, Z is the nucleic acid;    -   and wherein the linkage between X³ and Z is a phosphate or        thiophosphate

The branching unit may comprise carbon. Preferably, the branching unitis carbon.

X³ may be selected from the group consisting of —CH₂—O—C₄H₈—,—CH₂—O—C₅H₁₀—, —CH₂—O—CH₁₂—, —CH₂—O—C₇H₁₄—, and —CH₂—O—C₈H₁₆—.Preferably, X³ is selected from the group consisting of —CH₂—O—C₄H₈—,—CH₂—O—CH₁₂— and —CH₂—O—C₈H₁₆.

For any of the above aspects, when P represents a modified phosphategroup, P can be represented by:

wherein Y¹ and Y² each independently represent ═O, ═S, —O—, —OH, —SH,—BH₃, —OCH₂CO₂, —OCH₂CO₂R^(x), —OCH₂C(S)OR^(x), and —OR^(x), whereinR^(x) represents C₁-C₆ alkyl and wherein

indicates attachment to the remainder of the compound.

By modified phosphate it is meant a phosphate group wherein one or moreof oxygens is replaced. Examples of modified phosphate groups includephosphorothioate, phosphoroselenates, borano phosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl oraryl phosphonates and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulphur. One, each or both non-linkingoxygens in the phosphate group can be independently any one of S, Se, B,C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of a linkingoxygen with nitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at a terminal oxygen. Replacement of thenon-linking oxygens with nitrogen is possible.

For example, Y¹ may represent —OH and Y² may represent ═O or ═S; or

Y¹ may represent —O— and Y² may represent ═O or ═S;

Y¹ may represent ═O and Y² may represent —CH₃, —SH, —OR^(x), or —BH₃

Y¹ may represent ═S and Y² may represent —CH₃, OR^(x) or —SH.

It will be understood by the skilled person that in certain instancesthere will be delocalisation between Y¹ and Y².

Preferably, the modified phosphate group is a thiophosphate group.Thiophosphate groups include bithiophosphate (i.e. where Y¹ represents═S and Y² represents —S—) and monothiophosphate (i.e. where Y¹represents —O— and Y² represents ═S, or where Y¹ represents ═O and Y²represents —S—). Preferably, P is a monothiophosphate. The inventorshave found that conjugates having thiophosphate groups in replacement ofphosphate groups have improved potency and duration of action in vivo.

P may also be an ethylphosphate (i.e. where Y¹ represents ═O and Y²represents OCH₂CH₃).

The saccharide may be selected to have an affinity for at least one typeof receptor on a target cell. In particular, the receptor is on thesurface of a mammalian liver cell, for example, the hepaticasialoglycoprotein receptor (ASGP-R).

For any of the above aspects, the saccharide may be selected fromN-acetyl with one or more of galactosamine, mannose, galactose, glucose,glucosamine and fructose. Preferably, the saccharide is two molecules ofN-acetyl galactosamine (GalNAc). The compounds disclosed herein may have3 ligands which are each preferably N-acetyl galactosamine.

“GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonlyreferred to in the literature as N-acetyl galactosamine. Reference to“GalNAc” or “N-acetyl galactosamine” includes both the β-form:2-(Acetylamino)-2-deoxy-β-D-galactopyranose and the α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose. In certain embodiments,both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably.Preferably, the compounds disclosed herein comprise the β-form,2-(Acetylamino)-2-deoxy-β-D-galactopyranose.

-   2-(Acetylamino)-2-deoxy-D-galactopyranose

-   2-(Acetylamino)-2-deoxy-β-D-galactopyranose

-   2-(Acetylamino)-2-deoxy-α-D-galactopyranose

For any of the above compounds of formula (III), X¹ may be(—CH₂—CH₂—O)(—CH₂)₂—. X1 may be (—CH₂—CH₂—O)₂(—CH₂)₂—. X¹ may be(—CH₂—CH₂—O)₃(—CH₂)₂—. Preferably, X¹ is (—CH₂—CH₂—O)₂(—CH₂)₂—.Alternatively, X¹ represents C₃-C₆ alkylene. X¹ may be propylene. X¹ maybe butylene. X¹ may be pentylene. X¹ may be hexylene. Preferably thealkyl is a linear alkylene.

In particular, X¹ may be butylene.

For compounds of formula (III), X² represents an alkylene ether offormula —C₃H₆—O—CH₂—i.e. C₃ alkoxy methylene, or —CH₂CH₂CH₂OCH₂—.

The disclosure provides a conjugated nucleic acid having one of thefollowing structures:

wherein Z is a nucleic acid as defined herein before or after.

Preferred is a nucleic acid having one of the following structures

wherein Z is a nucleic acid as defined herein before or after, but anucleic acid with the first of these two structures is particularlypreferred.

The disclosure provides, as another aspect, a nucleic acid forinhibiting expression of TMPRSS6 in a cell, comprising at least oneduplex region that comprises at least a portion of a first strand and atleast a portion of a second strand that is at least partiallycomplementary to the first strand, wherein said first strand is at leastpartially complementary to at least a portion of a RNA transcribed fromthe TMPRSS6 gene, wherein said first strand comprises a nucleotidesequence selected from the following sequences: SEQ ID NOs: 317, 319,321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 353,356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 410,412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438,440, or 442,

or selected from SEQ ID Nos 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 67, 69, 71, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 85, 87,88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 225, 227, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,265, 267, 269, 270, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295, 297, 299, 301, 309, 311, 312, 313, 314, or 315,

such as selected from SEQ ID nos 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, or 215, wherein the nucleic acid is conjugated indirectly ordirectly to a ligand via a linker.

The second strand may comprise a nucleotide sequence of SEQ ID NO 318,320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,357, 359. 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411,413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439,441, or 443,

or selected from SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 68, 70, 72, 75, 84, 86, 89, 100, 101, 102, 103, 104, 105,106, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,226, 228, 230, 232, 234, 236, 238, 240, 242, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 310, 312, 314, or 316,

such as selected from SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198; 200, 202, 204, 206, 208, 210,212, 214, or 216.

The nucleic acid may be conjugated to a ligand as herein described.

The nucleotides of the first and/or second strand may be modified, asherein described.

Preferably, the nucleic acid comprises SEQ ID NO:17 and SEQ ID NO:18conjugated to a ligand of formula I (as set out above), particularly ofthe formula shown in FIG. 8a , and wherein the first strand is modifiedwith a 2′OMe modification on the odd numbered nucleotides, and modifiedwith a 2′F on the even numbered nucleotides, and the second strand ismodified with a 2′OMe on the even numbered nucleotides and modified witha 2′F on the odd numbered nucleotides.

More preferably, the nucleic acid comprises SEQ ID NO:17 and SEQ IDNO:18, wherein the nucleic acid is conjugated to a ligand of formula I(as set out above), and furthermore wherein the nucleic acid has amodification pattern as shown below which is an extract of Table 1 asherein provided.

SEQ ID 5′aaccagaagaagca 6273646282647284546 NO: 17 gguga 3′ SEQ ID5′ucaccugcuucuuc 1727354715351718451 NO: 18 ugguu 3′

wherein the specific modifications are depicted by numbers

1=2′F-dU,

2=2′F-dA,

3=2′F-dC,

4=2′F-dG,

5=2′-OMe-rU;

6=2′-OMe-rA;

7=2′-OMe-rC;

8=2′-OMe-rG.

The ligand may comprise GalNAc and FIG. 8a or FIG. 8b or FIG. 8c furtherillustrate the present invention.

Other preferred nucleic acids are listed above and below.

A cleavable linking group is a linker which is stable outside the cellbut is cleaved upon entry into a target cell. Cleavage releases the twoparts the linker is holding together.

In a preferred embodiment, the nucleic acid disclosed herein comprises acleavable linking group that is cleaved at least 10 times or more,preferably at least 100-fold faster in a target cell or under a firstreference condition (which can, for example, be selected to mimic orrepresent intracellular conditions) than in the blood of a subject, orunder a second reference condition (which can, for example, be selectedto mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g. pH,redox potential, or the presence of degradative molecules. Degradativemolecules include oxidative or reductive enzymes, reductive agents (suchas mercaptans), esterases, endosomes or agents than can create an acidicenvironment, enzymes that can hydrolyze or degrade an acid cleavablelinking group by acting as a general acid, peptidases, and phosphatases.

A cleavable linking group may be a disulphide bond, which is susceptibleto pH.

A linker may include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the target cell. For example, a linker thatincludes an ester group is preferred when a liver cell is the target.Linkers that contain peptide bonds can be used when targeting cells richin peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. In preferred embodiments, useful candidate compoundsare cleaved at least 2, 4, 10 or 100 times faster in the cell (or underin vitro conditions selected to mimic intracellular conditions) ascompared to blood or serum (or under in vitro conditions selected tomimic extracellular conditions).

In one aspect, the cleavable linking group may be a redox cleavablelinking group. The redox cleavable linking group may be a disulphidelinking group.

In one aspect, the linking group may be a phosphate-based cleavablelinking group.

Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferredembodiment is —O—P(O)(OH)—O—.

In one aspect, the cleavable linking group may be an acid cleavablelinking group. Preferably the acid cleavable linking group are cleavedin environments where the pH is 6.5 or lower, or are cleaved by agentssuch as enzymes that can act as a general acid. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—; C(O)O, or —OC(O). A preferred embodiment is alinking group where the carbon attached to the oxygen of the ester (thealkoxy group) is an aryl group, substituted alkyl group, or tertiaryalkyl group such as dimethyl pentyl or t-butyl.

In one embodiment, the cleavable linking group may be an ester-basedcleavable linking group. Examples of ester-based cleavable linkinggroups include but are not limited to esters of alkylene, alkenylene andalkynylene groups.

In one embodiment, the cleavable linking group may be a peptide-basedcleavable linking group. Peptide-based cleavable linking groups arepeptide bonds formed between amino acids to yield oligopeptides (e.g.,dipeptides, tripeptides etc.) and polypeptides. The peptide basedcleavage group is generally limited to the peptide bond (i.e., the amidebond) formed between amino acids yielding peptides and proteins and doesnot include the entire amide functional group. Peptide-based cleavablelinking groups have the general formula —NHCHR^(A)C(O)NHCHR^(B)C(O)—,where RA and RB are the R groups of the two adjacent amino acids.

The nucleic acid as described herein may be formulated with a lipid inthe form of a liposome. Such a formulation may be described in the artas a lipoplex. The formulation with a lipid/liposome may be used toassist with delivery of the nucleic acid disclosed herein to the targetcells. The lipid delivery system herein described may be used as analternative to a conjugated ligand. The modifications herein describedmay be present when using the nucleic acid disclosed herein with a lipiddelivery system or with a ligand conjugate delivery system.

Such a lipoplex may comprise a lipid formulation comprising:

-   -   i) a cationic lipid, or a pharmaceutically acceptable salt        thereof;    -   ii) a steroid;    -   iii) a phosphatidylethanolamine phospholipid;    -   iv) a PEGylated lipid.

The cationic lipid may be an amino cationic lipid.

The cationic lipid may have the formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

X represents O, S or NH;

R¹ and R² each independently represents a C₄-C₂₂ linear or branchedalkyl chain or a C₄-C₂₂ linear or branched alkenyl chain with one ormore double bonds, wherein the alkyl or alkenyl chain optionallycontains an intervening ester, amide or disulfide;

when X represents S or NH, R³ and R⁴ each independently representhydrogen, methyl, ethyl, a mono- or polyamine moiety, or R³ and R⁴together form a heterocyclyl ring;

when X represents O, R³ and R⁴ each independently represent hydrogen,methyl, ethyl, a mono- or polyamine moiety, or R³ and R⁴ together form aheterocyclyl ring, or R³ represents hydrogen and R⁴ representsC(NH)(NH₂).

The cationic lipid may have the formula (V):

or a pharmaceutically acceptable salt thereof.

The cationic lipid may have the formula (VI):

or a pharmaceutically acceptable salt thereof.

The content of the cationic lipid component may be from about 55 mol %to about 65 mol % of the overall lipid content of the formulation. Inparticular, the cationic lipid component is about 59 mol % of theoverall lipid content of the formulation.

The formulations further comprise a steroid. The steroid may becholesterol. The content of the steroid may be from about 26 mol % toabout 35 mol % of the overall lipid content of the lipid formulation.More particularly, the content of steroid may be about 30 mol % of theoverall lipid content of the lipid formulation.

The phosphatidylethanolamine phospholipid may be selected from groupconsisting of 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE),1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE),1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE),1,2-Disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE) and1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE). Thecontent of the phospholipid may be about 10 mol % of the overall lipidcontent of the formulation.

The PEGylated lipid may be selected from the group consisting of1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG) andC16-Ceramide-PEG. The content of the PEGylated lipid may be about 1 to 5mol % of the overall lipid content of the formulation.

The content of the cationic lipid component in the formulation may befrom about 55 mol % to about 65 mol % of the overall lipid content ofthe lipid formulation, preferably about 59 mol % of the overall lipidcontent of the lipid formulation.

The formulation may have a molar ratio of the components ofi):ii):iii):iv) selected from 55:34:10:1; 56:33:10:1; 57:32:10:1;58:31:10:1; 59:30:10:1; 60:29:10:1; 61:28:10:1; 62:27:10:1; 63:26:10:1;64:25:10:1; and 65:24:10:1.

The formulation may comprise a cationic lipid having the structure

a steroid having the structure

a phosphatidylethanolamine phospholipid having the structure

And a PEGylated lipid having the structure

Neutral liposome compositions may be formed from, for example,dimyristoyl phosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions may be formedfrom dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomesmay be formed primarily from dioleoyl phosphatidylethanolamine (DOPE).Another type of liposomal composition may be formed fromphosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.Another type is formed from mixtures of phospholipid and/orphosphatidylcholine and/or cholesterol.

A positively charged synthetic cationic lipid,N—[I-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells. DOTMA analogues can also be used to formliposomes.

Derivatives and analogues of lipids described herein may also be used toform liposomes.

A liposome containing a nucleic acid can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The nucleicacid preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the nucleicacid and condense around the nucleic acid to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of nucleic acid.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also be adjusted to favourcondensation.

Nucleic acid formulations may include a surfactant. In one embodiment,the nucleic acid is formulated as an emulsion that includes asurfactant.

A surfactant that is not ionized is a non-ionic surfactant. Examplesinclude non-ionic esters, such as ethylene glycol esters, propyleneglycol esters, glyceryl esters etc., nonionic alkanolamides, and etherssuch as fatty alcohol ethoxylates, propoxylated alcohols, andethoxylated/propoxylated block polymers.

A surfactant that carries a negative charge when dissolved or dispersedin water is an anionic surfactant. Examples include carboxylates, suchas soaps, acyl lactylates, acyl amides of amino acids, esters ofsulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates,sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyltaurates and sulfosuccinates, and phosphates.

A surfactant that carries a positive charge when dissolved or dispersedin water is a cationic surfactant. Examples include quaternary ammoniumsalts and ethoxylated amines.

A surfactant that has the ability to carry either a positive or negativecharge is an amphoteric surfactant. Examples include acrylic acidderivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

“Micelles” are defined herein as a particular type of molecular assemblyin which amphipathic molecules are arranged in a spherical structuresuch that all the hydrophobic portions of the molecules are directedinward, leaving the hydrophilic portions in contact with the surroundingaqueous phase. The converse arrangement exists if the environment ishydrophobic. A micelle may be formed by mixing an aqueous solution ofthe nucleic acid, an alkali metal alkyl sulphate, and at least onemicelle forming compound.

Exemplary micelle forming compounds include lecithin, hyaluronic acid,pharmaceutically acceptable salts of hyaluronic acid, glycolic acid,lactic acid, chamomile extract, cucumber extract, oleic acid, linoleicacid, linolenic acid, monoolein, monooleates, monolaurates, borage oil,evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine andpharmaceutically acceptable salts thereof, glycerin, polyglycerin,lysine, polylysine, triolein, polyoxyethylene ethers and analoguesthereof, polidocanol alkyl ethers and analogues thereof,chenodeoxycholate, deoxycholate, and mixtures thereof.

Phenol and/or m-cresol may be added to the mixed micellar composition toact as a stabiliser and preservative. An isotonic agent such asglycerine may as be added.

A nucleic acid preparation may be incorporated into a particle such as amicroparticle.

Microparticles can be produced by spray-drying, lyophilisation,evaporation, fluid bed drying, vacuum drying, or a combination of thesemethods.

Iron Chelators

Iron chelation therapy is used to reduce iron overload. Iron is removedfrom different tissues at different rates. The aim of chelation therapyis to prevent the accumulation of iron excess and its complication suchas hepatic endocrinological and cardiac dysfunction. Several ironchelators have been designed to excrete tissue iron through the urine orfaeces by forming complexes.

1. Deferoxamine: This is a clinically approved chelator, which iseffective for long term iron chelation therapy in β-thalassaemia andother diseases with iron overload. Because of its short plasmahalf-life, it is given by continuous intravenous or subcutaneousinfusion in order to be effective.

2. Deferiprone: This is an oral chelator usually taken 3 times a day

3. Deferasirox (Exjade): This is another oral iron chelator with anextended half-life. It is prescribed for daily use.

4. Deferasirox (Jadenu): This is a film coated tablet, with aformulation different from Deferasirox (Exjade). The advantage is, thatit can be taken together with a light meal.

The preferred iron chelator in the context of this disclosure isdeferiprone.

The different chelators have a different spectrum of side effects, i.e.injection site reactions by Deferoxamine, renal insufficiencies byDeferoxamine, and Deferasirox, athralgia and neutropenia withDeferiprone. Gastrointestinal disturbances are side effects reported byall classes of chelators. Patient compliance to chelation therapycontinues to be an issue, but has improved with the availability or ironchelators that can be taken orally (Chalmers A W, Shammo J M. 2016.‘Evaluation of a new tablet formulation of deferasirox to reduce chroniciron overload after long-term blood transfusions’, Ther Clin Risk Manag,12: 201-8; and Mobarra N, Shanaki M, Ehteram H, Nasiri H, Sahmani M,Saeidi M, Goudarzi M, Pourkarim H, Azad M. 2016. ‘A Review on IronChelators in Treatment of Iron Overload Syndromes’, Int J Hematol OncolStem Cell Res, 10: 239-47).

One advantage of using the present combination of a chelator and ansiRNA that inhibits TMPRSS6, especially for hemochromatosis treatment isthat the combined treatment allows to reduce iron liver levels quicklyand efficiently (see example 47). After the initial combinationtreatment, the amount of chelator doses can be drastically reduced andtreatment can continue essentially only with the siRNA, supplementedwith the chelator as and when is necessary. This is highly beneficialfor patients due to the side effects they experience from ironchelators. Accordingly, one aspect of the present invention is acombination, a combination for use, a nucleic acid for use, a kit, acomposition, a composition for use or a method of treatment wherein anucleic acid and an iron chelator as described herein are administeredin combination in a first phase of the treatment and wherein the dosageof iron chelator is strongly reduced in a second phase of the treatmentcompared to the first phase. Strongly reduced in this context meansreduced by at least 50%, preferably at least 60%, more preferably atleast 70% and most preferably at least 80% between comparable amounts oftime of the first and second phase. The duration of the first phase ispreferably three weeks or less, more preferably two weeks or less.

The present invention also provides pharmaceutical compositionscomprising a nucleic acid or conjugated nucleic acid of the disclosure,in combination with one or more iron chelators. The pharmaceuticalcompositions may be used as medicaments or as diagnostic agents, aloneor in combination with other agents. For example, a nucleic acid orconjugated nucleic acid and/or iron chelator can be combined with adelivery vehicle (e.g., liposomes) and excipients, such as carriers,diluents. Other agents such as preservatives and stabilizers can also beadded. Methods for the delivery of nucleic acids are known in the artand within the knowledge of the person skilled in the art.

The nucleic acid or conjugated nucleic acid and the one or more ironchelators can be administered separately, sequentially orsimultaneously, e.g., as a combined unit dose.

It should be noted that a combination in the context of this disclosuredoes not necessarily mean that two elements (such as the nucleic acidand the chelator) are combined in a single composition. Rather itpreferably means that the two elements, such as the nucleic acid and thechelator are for combined administration, which can occur at the sametime for both elements or within a short period of time of each other. Ashort period of time is preferably one week, but more preferably 6 days,more preferably 5 days, more preferably 4 days, more preferably 3 days,more preferably 2 days, more preferably 1 day and most preferably lessthan one day, such as 12 hours, 6 hours, 4 hours, 2 hours or 1 hour.

A nucleic acid or conjugated nucleic acid of the disclosure, incombination with one or more iron chelators can also be administered incombination with other therapeutic compounds, either administratedseparately or simultaneously, e.g., as a combined unit dose. Theinvention also includes a pharmaceutical composition comprising anucleic acid or conjugated nucleic acid of the disclosure, incombination with one or more iron chelators, in aphysiologically/pharmaceutically acceptable excipient, such as astabilizer, preservative, diluent, buffer, and the like. The inventionalso includes a pharmaceutical composition comprising a nucleic acid orconjugated nucleic acid in a physiologically/pharmaceutically acceptableexcipient, such as a stabilizer, preservative, diluent, buffer, and thelike, and a pharmaceutical composition comprising one or more ironchelators in a physiologically/pharmaceutically acceptable excipient,such as a stabilizer, preservative, diluent, buffer, and the like.

The pharmaceutical composition may be specially formulated foradministration in solid or liquid form. The composition may beformulated for oral administration, parenteral administration(including, for example, subcutaneous, intramuscular, intravenous, orepidural injection), topical application, intravaginal or intrarectaladministration, sublingual administration, ocular administration,transdermal administration, or nasal administration. Delivery usingsubcutaneous or intravenous methods are preferred.

In one embodiment, a unit dose may contain between about 0.01 mg/kg andabout 100 mg/kg body weight of nucleic acid or conjugated nucleic acid.Alternatively, the dose can be from 10 mg/kg to 25 mg/kg body weight, or1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight,or 0.1 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 1 mg/kg bodyweight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kgbody weight. Dosage levels may also be calculated via other parameterssuch as, e.g., body surface area.

The pharmaceutical composition may be a sterile injectable aqueoussuspension or solution, or in a lyophilized form. In one embodiment, thepharmaceutical composition may comprise lyophilized lipoplexes or anaqueous suspension of lipoplexes. The lipoplexes preferably comprises anucleic acid disclosed herein. Such lipoplexes may be used to deliverthe nucleic acid disclosed hereinto a target cell either in vitro or invivo.

The pharmaceutical compositions and medicaments of the present inventionmay be administered to a mammalian subject in a pharmaceuticallyeffective dose. The mammal may be selected from a human, a non-humanprimate, a simian or prosimian, a dog, a cat, a horse, cattle, a pig, agoat, a sheep, a mouse, a rat, a hamster, a hedgehog and a guinea pig,or other species of relevance. On this basis, the wording “TMPRSS6” asused herein denotes nucleic acid or protein in any of the abovementioned species, but preferably this wording denotes human nucleicacids or proteins.

A further aspect of the invention relates to a nucleic acid disclosedherein or the pharmaceutical composition comprising the nucleic aciddisclosed herein, in combination with one or more iron chelators, foruse in the treatment or prevention of a disease or disorder. The diseaseor disorder may be selected from the group comprising hemochromatosis,porphyria cutanea tarda and blood disorders, such as β-thalassemias orsickle cell disease, congenital dyserythropoietic anemia, marrow failuresyndrome, myelodysplasia and transfusional iron overload. The disordermay be associated with iron overload and the disorder associated withiron overload may be Parkinson's Disease, Alzheimer's Disease orFriedreich's Ataxia. Preferably, the disorder is hemochromatosis.

In particular, preferred aspects of the invention include: Any nucleicacid as disclosed herein in combination with one or more iron chelatorsfor use in one or more or all of:

(i) treatment of anemia, optionally suitably determined by an increasein haemoglobin or haematocrit or by decrease in reticulocytes asdisclosed by the methods herein; and/or

(ii) reduction of splenomegaly, optionally determined by a reduction inspleen size by weight as disclosed by the methods herein: and/or

(iii) amelioration of ineffective erythropoiesis in spleen, optionallydetermined by reduction in relative proportion of immature erythroidcells in spleen determined by FACS analysis, as described herein; and/or

(iv) Improvement of red blood cell maturation/erythropoiesis in the bonemarrow, optionally determined by a decrease in relative proportion oferythroid progenitor cells and increase in enucleated erythroid cellsdetermined by FACS analysis, as described herein;

(v) treatment of hemochromatosis;

(vi) treatment of iron overload.

The invention includes a pharmaceutical composition comprising one ormore RNAi molecule disclosed herein in combination with one or more ironchelators in a physiologically/pharmaceutically acceptable excipient,such as a stabiliser, preservative, diluent, buffer and the like.

The invention includes a pharmaceutical composition comprising one ormore RNAi molecule disclosed herein in aphysiologically/pharmaceutically acceptable excipient, such as astabiliser, preservative, diluent, buffer and the like, and apharmaceutical composition comprising one or more iron chelators in aphysiologically/pharmaceutically acceptable excipient, such as astabiliser, preservative, diluent, buffer and the like.

The pharmaceutical composition may be a sterile injectable aqueoussuspension or solution, or in a lyophilised form.

Pharmaceutically acceptable compositions may comprise atherapeutically-effective amount of one or more nucleic acid(s) in anyembodiment according to the invention, taken alone or formulated withone or more pharmaceutically acceptable carriers, excipient and/ordiluents.

Examples of materials which can serve as pharmaceutically-acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

Stabilisers may be agents that stabilise the nucleic acid agent, forexample a protein that can complex with the nucleic acid, chelators(e.g. EDTA), salts, RNAse inhibitors, and DNAse inhibitors.

In some cases it is desirable to slow the absorption of the drug fromsubcutaneous or intramuscular injection in order to prolong the effectof a drug. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

The nucleic acid described herein may be capable of inhibiting theexpression of TMPRSS6. The nucleic acid described herein may be capableof partially inhibiting the expression of TMPRSS6 in a cell. Inhibitionmay be complete, i.e. 0% of the expression level of TMPRSS6 expressionin the absence of the nucleic acid disclosed herein. Inhibition ofTMPRSS6 expression may be partial, i.e. it may be 15%, 20%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of TMRPSS6 expression in theabsence of a nucleic acid disclosed herein. Inhibition may last 2 weeks,3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 11weeks, 12 weeks, 13 weeks, 14 weeks, or up to 6 months, when used in asubject, such as a human subject. A nucleic acid or conjugated nucleicacid disclosed herein, or compositions including the same, may be foruse in a regimen comprising treatments once or twice weekly, every week,every two weeks, every three weeks, every four weeks, every five weeks,every six weeks, every seven weeks, or every eight weeks, or in regimenswith varying dosing frequency such as combinations of thebefore-mentioned intervals. The nucleic acid may be for usesubcutaneously, intravenously or using any other application routes suchas oral, rectal or intraperitoneal.

In cells and/or subjects treated with or receiving the nucleic aciddisclosed herein, the TMPRSS6 expression may be inhibited compared tountreated cells and/or subjects by a range from 15% up to 100% but atleast about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 100%. The level of inhibition may allow treatment of adisease associated with TMPRSS6 expression or overexpression, or mayallow further investigation into the functions of the TMPRSS6 geneproduct.

Hepcidin gene expression in cells and/or subjects treated with a nucleicacid or conjugated nucleic acid disclosed herein may be increasedcompared to untreated cells and/or subjects by at least about 1.2 fold,about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, or about5-fold, due to the complete or partial inhibition of TMPRSS6 geneexpression. This may lead to serum hepcidin concentration in subjectstreated a nucleic acid or conjugated nucleic acid disclosed herein maybe increased compared to untreated subjects by at least about 10%, about25%, about 50%, about 100%, about 150%, about 200%, about 250%, about300%, or about 500%.

Serum or plasma iron concentration in subjects treated with a nucleicacid or conjugated nucleic acid disclosed herein may be decreasedcompared to untreated subjects by at least about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%. Transferrinsaturation is a medical laboratory test that measures the amount oftransferrin that is bound to iron. Transferrin saturation, measured as apercentage, is a medical laboratory value that measures the value ofserum iron divided by the total transferrin iron-binding capacity.Transferrin saturation in subjects treated with a nucleic acid orconjugated nucleic acid disclosed herein may be decreased compared tountreated subjects by at least about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%.

A further aspect of the invention relates to a nucleic acid disclosedherein in combination with one or more iron chelators in the manufactureof a medicament for treating or preventing a disease or disorder, suchas disease or disorder as listed above.

Also included in the invention is a method of treating or preventing adisease or disorder, such as those listed above, comprisingadministration of a pharmaceutical composition comprising a nucleic acidor conjugated nucleic acid disclosed herein in combination with one ormore iron chelators, to an individual in need of treatment. Thecomposition may be administered twice every week, once every week, everytwo weeks, every three weeks, every four weeks, every five weeks, everysix weeks, every seven weeks, or every eight weeks. The nucleic acid orconjugated nucleic acid in combination with one or more iron chelatorsmay be for use subcutaneously or intravenously or other applicationroutes such as oral, rectal or intraperitoneal. The nucleic acid andchelator can be administered as a single composition that comprises bothor in different compositions that are administered separately, either bythe same route or by a different route, at the same or different dosage,and at the same or different dosage frequencies.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of a nucleic acid agent. The maintenance dose ordoses can be the same or lower than the initial dose, e.g., one-halfless of the initial dose. The maintenance doses are, for example,administered no more than once every 2, 5, 10, or 30 days. The treatmentregimen may last for a period of time which will vary depending upon thenature of the particular disease, its severity and the overall conditionof the patient. The subject may be administered a chelator incombination with the siRNA either throughout the treatment with thesiRNA or only for part of the treatment with the siRNA. The dosage ofthe chelator that is administered in combination with the siRNA may beadapted to the particular need of the subject. The need of the subjectmay be determined for example based on the measurement of liver ironlevels or other markers that indicate the need for a combined treatment.

In one embodiment, the composition includes a plurality of nucleic acidagent species. In another embodiment, the nucleic acid agent species hassequences that are non-overlapping and non-adjacent to another specieswith respect to a naturally occurring target sequence. In anotherembodiment, the plurality of nucleic acid agent species is specific fordifferent naturally occurring target genes. In another embodiment, thenucleic acid agent is allele specific.

A nucleic acid or conjugated nucleic acid of the disclosure, incombination with one or more iron chelators, can also be administered orfor use in combination with other therapeutic compounds, eitheradministered separately or simultaneously, e.g. as a combined unit dose.

The nucleic acid or conjugated nucleic acid of the present disclosure,in combination with one or more iron chelators, can be produced usingroutine methods in the art including chemical synthesis or expressingthe nucleic acid either in vitro (e.g., run off transcription) or invivo. For example, using solid phase chemical synthesis or using anucleic acid-based expression vector including viral derivates orpartially or completely synthetic expression systems. In one embodiment,the expression vector can be used to produce the nucleic acid disclosedherein in vitro, within an intermediate host organism or cell type,within an intermediate or the final organism or within the desiredtarget cell. Methods for the production (synthesis or enzymatictranscription) of the nucleic acid described herein are known to personsskilled in the art.

In further embodiments of the invention, the invention relates to anynucleic acid, conjugated nucleic acid, nucleic acid for use, method,combination, combination for use, kit, composition or use according toany disclosure herein, wherein the nucleic acid comprises avinyl-(E)-phosphonate modification, such as a 5′ vinyl-(E)-phosphonatemodification, preferably a 5′ vinyl-(E)-phosphonate modification incombination with a 2′-F modification at the second position of the firststrand.

In further embodiments of the invention, the invention relates to anynucleic acid, conjugated nucleic acid, nucleic acid for use, method,combination, combination for use, kit, composition or use according toany disclosure herein, wherein the terminal nucleotide at the 3′ end ofat least one of the first strand and the second strand is an invertednucleotide and is attached to the adjacent nucleotide via the 3′ carbonof the terminal nucleotide and the 3′ carbon of the adjacent nucleotideand/or the terminal nucleotide at the 5′ end of at least one of thefirst strand and the second strand is an inverted nucleotide and isattached to the adjacent nucleotide via the 5′ carbon of the terminalnucleotide and the 5′ carbon of the adjacent nucleotide,

optionally wherein

-   -   a. the 3′ and/or 5′ inverted nucleotide of the first and/or        second strand is attached to the adjacent nucleotide via a        phosphate group by way of a phosphodiester linkage; or    -   b. the 3′ and/or 5′ inverted nucleotide of the first and/or        second strand is attached to the adjacent nucleotide via a        phosphorothioate group or    -   c. the 3′ and/or 5′ inverted nucleotide of the first and/or        second strand is attached to the adjacent nucleotide via a        phosphorodithioate group.

In further embodiments of the invention, the invention relates to anynucleic acid, conjugated nucleic acid, nucleic acid for use, method,combination, combination for use, kit, composition or use according toany disclosure herein, wherein the nucleic acid comprises aphosphorodithioate linkage,

optionally wherein the linkage is between the 2 most 5′ nucleotidesand/or the 2 most 3′ nucleotides of the second strand, and/or

optionally wherein the nucleic acid additionally does not comprise anyinternal phosphorothioate linkages.

In further embodiments of the invention, the invention relates to anucleic acid sequence disclosed herein, in combination with one or moreiron chelators, wherein the first strand and/or the second strand of thenucleic acid comprises at least a first and a second type of modifiedribonucleotide, and wherein there is more of the first type of modifiedribonucleotide than the second type of modified nucleotide (for examplemore of a 2′ O methyl modification than a 2′ fluoro modification),either as counted on each strand individually, or across both the firstand second strand.

For the avoidance of doubt the nucleic acid may have more than two typesof modification.

Another embodiment of the invention relates to any nucleic acid sequencedisclosed herein, in combination with one or more iron chelators,wherein greater than 50% of the nucleotides of the first and/or secondstrand comprise a 2′ O-methyl modification, such as greater than 55%,60%, 65%, 70%, 75%, 80%, or 85%, or more, of the first and/or secondstrand comprise a 2′ O-methyl modification, preferably measured as apercentage of the total nucleotides of both the first and secondstrands.

Another embodiment of the invention relates to any nucleic acid sequencedisclosed herein, in combination with one or more iron chelators,wherein greater than 50% of the nucleotides of the first and/or secondstrand comprise a naturally occurring RNA modification, such as whereingreater than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the firstand/or second strands comprise such a modification, preferably measuredas a percentage of the total nucleotides of both the first and secondstrands. Suitable naturally occurring modifications include, as well as2-0′ methyl, other 2′ sugar modifications, in particular a 2′-Hmodification resulting in a DNA nucleotide.

Another embodiment of the invention relates to any nucleic acid sequencedisclosed herein, in combination with one or more iron chelators,comprising no more than 20%, such as no more than 15%, such as no morethan 10%, of nucleotides which have 2′ modifications that are not 2′ 0methyl modifications on the first and/or second strand, preferably as apercentage of the total nucleotides of both the first and secondstrands.

Another embodiment of the invention relates to any nucleic acid sequencedisclosed herein, in combination with one or more iron chelators,comprising no more than 20%, (such as no more than 15% or no more than10%) of 2′ fluoro modifications on the first and/or second strand,preferably as a percentage of the total nucleotides of both strands.

Another embodiment of the invention relates to a nucleic acid forinhibiting expression of TMPRSS6 in combination with one or more ironchelators, comprising at least one duplex region that comprises at leasta portion of a first strand and at least a portion of a second strandthat is at least partially complementary to the first strand, whereinsaid first strand is at least partially complementary to at least aportion of RNA transcribed from the TMPRSS6 gene, wherein said firststrand comprises a nucleotide sequence of HC18A as below; and optionallywherein said second strand comprises the nucleotide sequence of HC18b asbelow:

TMPRSS6-hc-18A UUUUCUCUUGGAGUCCUCA TMPRSS6-hc-18B UGAGGACUCCAAGAGAAAA

Optionally the nucleic acid sequences are either TMPRSS6-hc-18A, or bothof TMPRSS6-hc-18A and 18B as below:

TMPRSS6-hc-18A mU (ps) fU (ps) mUfUmCfUmCfUmUfGmGfAmGfUmCfCmU(ps) fC (ps) mA TMPRSS6-hc-18BfUmGfAmGfGmAfCmUfCmCfAmAfGmAfGmAfA (ps) mA (ps) fA

Optionally the nucleic acid sequences are either or both of thesequences listed below.

TMPRSS6-hc-18A mU (ps) fU (ps) mUfUmCfUmCfUmUfGmGfAmGfUmCfCmU (ps)fC (ps) mA TMPRSS6-hc-18BGN3 - fUmGfAmGfGmAfCmUfCmCfAmAfGmAfGmAfA (ps) mA (ps) fA

All sequences are listed 5′-3′. A nucleic acid for inhibiting expressionof TMPRSS6, comprising at least one duplex region that comprises atleast a portion of a first strand and at least a portion of a secondstrand that is at least partially complementary to the first strand,wherein said first strand is at least partially complementary to atleast a portion of RNA transcribed from the TMPRSS6 gene, wherein saidfirst strand comprises a nucleotide sequence of HC23A as below; andoptionally wherein said second strand comprises the nucleotide sequenceof HC23B as below:

TMPRSS6-hc-23A CUGUUCUGGAUCGUCCACU TMPRSS6-hc-23B AGUGGACGAUCCAGAACAG

Optionally the nucleic acid sequences are either TMPRSS6-hc-23A, or bothof TMPRSS6-hc-23A and 23B as below:

TMPRSS6-hc-23A mC (ps) fU (ps) mGfUmUfCmUfGmGfAmUfCmGfUmCfCmA(ps) fC (ps) mU TMPRSS6-hc-23BfAmGfUmGfGmAfCmGfAmUfCmCfAmGfAmAfC (ps) mA (ps) fG

Optionally the nucleic acid sequences are either or both of thesequences listed below.

TMPRSS6-hc-23A mC (ps) fU (ps) mGfUmUfCmUfGmGfAmUfCmGfUmCfCmA(ps) fC (ps) mU TMPRSS6-hc-23BGN3 - fAmGfUmGfGmAfCmGfAmUfCmCfAmGfAmAfC (ps) mA (ps) fG

Another embodiment of the invention is a nucleic acid for inhibitingexpression of TMPRSS in combination with one or more iron chelators in acell, comprising at least one duplex region that comprises at least aportion of a first strand and at least a portion of a second strand thatis at least partially complementary to the first strand, wherein saidfirst strand is at least partially complementary to at least a portionof RNA transcribed from the TMPRSS gene, wherein the expression ofTMPRSS in a cell is reduced to levels which are at least 15% lower thanexpression levels observed in same test conditions but in the absence ofthe nucleic acid or conjugated nucleic acid or in the presence of anon-silencing control.

The nucleic acid may be any disclosed herein.

Certain Preferred Embodiments

1. A combination comprising a nucleic acid for inhibiting expression ofTMPRSS6 and one or more iron chelators,

wherein the nucleic acid comprises at least one duplex region thatcomprises at least a portion of a first strand and at least a portion ofa second strand that is at least partially complementary to the firststrand, wherein said first strand is at least partially complementary toat least a portion of RNA transcribed from the TMPRSS6 gene, whereinsaid first strand comprises a nucleotide sequence selected from thefollowing sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329,331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394,396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422,424, 426, 428, 430, 432, 434, 436, 438, 440, or 442, preferably SEQ IDNO: 333.

2. A combination for use in the treatment of hemochromatosis, porphyriacutanea tarda, blood disorders, such as β-thalassemia or sickle celldisease, congenital dyserythropoietic anemia, marrow failure syndromes,myelodysplasia, transfusional iron overload, a disorder associated withiron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich'sAtaxia associated with iron overload, and infections and non-relapserelated mortality associated with bone marrow transplantation,preferably for the treatment of hemochromatosis,

the combination (or composition) comprising a nucleic acid forinhibiting expression of TMPRSS6 and one or more iron chelators, whereinthe nucleic acid comprises at least one duplex region that comprises atleast a portion of a first strand and at least a portion of a secondstrand that is at least partially complementary to the first strand,wherein said first strand is at least partially complementary to atleast a portion of RNA transcribed from the TMPRSS6 gene, wherein saidfirst strand comprises a nucleotide sequence selected from the followingsequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335,337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398,400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426,428, 430, 432, 434, 436, 438, 440, or 442, preferably SEQ ID NO: 333.

3. A nucleic acid for use as a medicament, preferably a nucleic acid foruse in the treatment of hemochromatosis, porphyria cutanea tarda, blooddisorders, such as β-thalassemia or sickle cell disease, congenitaldyserythropoietic anemia, marrow failure syndromes, myelodysplasia,transfusional iron overload, a disorder associated with iron overload,Parkinson's Disease, Alzheimer's Disease or Friedreich's Ataxiaassociated with iron overload, and infections and non-relapse relatedmortality associated with bone marrow transplantation, preferably forthe treatment of hemochromatosis,

wherein the nucleic acid inhibits expression of TMPRSS6, wherein thenucleic acid comprises at least one duplex region that comprises atleast a portion of a first strand and at least a portion of a secondstrand that is at least partially complementary to the first strand,wherein said first strand is at least partially complementary to atleast a portion of RNA transcribed from the TMPRSS6 gene, wherein saidfirst strand comprises a nucleotide sequence selected from the followingsequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335,337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398,400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426,428, 430, 432, 434, 436, 438, 440, or 442, preferably SEQ ID NO: 333,

wherein the nucleic acid is administered with one or more ironchelators.

4. A kit comprising a nucleic acid for inhibiting expression of TMPRSS6and one or more iron chelators,

wherein the nucleic acid comprises at least one duplex region thatcomprises at least a portion of a first strand and at least a portion ofa second strand that is at least partially complementary to the firststrand, wherein said first strand is at least partially complementary toat least a portion of RNA transcribed from the TMPRSS6 gene, whereinsaid first strand comprises a nucleotide sequence selected from thefollowing sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329,331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394,396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422,424, 426, 428, 430, 432, 434, 436, 438, 440, or 442, preferably SEQ IDNO: 333.

5. A pharmaceutical composition comprising a nucleic acid for inhibitingexpression of TMPRSS6 and one or more iron chelators,

wherein the nucleic acid comprises at least one duplex region thatcomprises at least a portion of a first strand and at least a portion ofa second strand that is at least partially complementary to the firststrand, wherein said first strand is at least partially complementary toat least a portion of RNA transcribed from the TMPRSS6 gene, whereinsaid first strand comprises a nucleotide sequence selected from thefollowing sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329,331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394,396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422,424, 426, 428, 430, 432, 434, 436, 438, 440, or 442, preferably SEQ IDNO: 333,

wherein the nucleic acid, iron chelator or both are combined with aphysiologically acceptable excipient.

6. A pharmaceutical composition for use in the treatment ofhemochromatosis, porphyria cutanea tarda, blood disorders, such asβ-thalassemia or sickle cell disease, congenital dyserythropoieticanemia, marrow failure syndromes, myelodysplasia, transfusional ironoverload, a disorder associated with iron overload, Parkinson's Disease,Alzheimer's Disease or Friedreich's Ataxia associated with ironoverload, and infections and non-relapse related mortality associatedwith bone marrow transplantation, preferably for the treatment ofhemochromatosis,

wherein the pharmaceutical composition comprises a nucleic acid forinhibiting expression of TMPRSS6 and one or more iron chelators, whereinthe nucleic acid comprises at least one duplex region that comprises atleast a portion of a first strand and at least a portion of a secondstrand that is at least partially complementary to the first strand,wherein said first strand is at least partially complementary to atleast a portion of RNA transcribed from the TMPRSS6 gene, wherein saidfirst strand comprises a nucleotide sequence selected from the followingsequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335,337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398,400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426,428, 430, 432, 434, 436, 438, 440, or 442, preferably SEQ ID NO: 333

wherein the nucleic acid, iron chelator or both are combined with aphysiologically acceptable excipient.

7. The combination of preferred embodiment 1, combination for use ofpreferred embodiment 2, nucleic acid for use of preferred embodiment 3,kit of preferred embodiment 4, composition of preferred embodiment 5 orcomposition for use of preferred embodiment 6, wherein the second strandcomprises a nucleotide sequence of SEQ ID NO: 334, 318, 320, 322, 324,326, 328, 330, 332, 336, 338, 340, 342, 344, 346, 357, 359. 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391,393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419,421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, or 443,preferably SEQ ID NO: 334.

8. The combination, combination for use, nucleic acid for use, kit,composition or composition for use according to any of the preferredembodiments 1-7, wherein the one or more iron chelators are selectedfrom deferoxamine, deferiprone, deferasirox (Exjade) and deferairox(Jadenu), optionally wherein the iron chelator is deferiprone.

9. The combination, combination for use, nucleic acid for use, kit,composition or composition for use according to any of the preferredembodiments 1-8, wherein one or more nucleotides on the first and/orsecond strand are modified, to form modified nucleotides. The nucleicacid may comprise a modification that is 2′-O-methyl (2′OMe) that may bea first modification, and a second modification that is 2′-F. Modifiednucleotides are evidently nucleotides that conserve there base identity(A, C, G, T, U) but have one or several modifications for example on thesugar, such as a modification of the 2′-OH to a 2′OMe or 2′F.

10. The combination, combination for use, or nucleic acid for useaccording to preferred embodiment 9, wherein said first strand comprisesa nucleotide sequence of SEQ ID NO:17, and wherein said second strandcomprises the nucleotide sequence of SEQ ID NO:18

SEQ ID 5′aaccagaagaagca 6273646282647284546 NO: 17 gguga 3′ SEQ ID5′ucaccugcuucuuc 1727354715351718451 NO: 18 ugguu 3′

wherein the specific modifications are depicted by the following numbers

1=2′F-dU,

2=2′F-dA,

3=2′F-dC,

4=2′F-dG,

5=2′-OMe-rU;

6=2′-OMe-rA;

7=2′-OMe-rC;

8=2′-OMe-rG.

11. A combination comprising a nucleic acid for inhibiting expression ofTMPRSS6 and deferiprone,

wherein the nucleic acid comprises at least one duplex region thatcomprises at least a portion of a first strand and at least a portion ofa second strand that is at least partially complementary to the firststrand, wherein said first strand is at least partially complementary toat least a portion of RNA transcribed from the TMPRSS6 gene, wherein thefirst strand comprises the nucleotide sequence of SEQ ID NO:17 and thesecond strand comprises the nucleotide sequence of SEQ ID NO:18.

12. A combination for use in the treatment of hemochromatosis, porphyriacutanea tarda, blood disorders, such as β-thalassemia or sickle celldisease, congenital dyserythropoietic anemia, marrow failure syndromes,myelodysplasia, transfusional iron overload, a disorder associated withiron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich'sAtaxia associated with iron overload, and infections and non-relapserelated mortality associated with bone marrow transplantation,preferably for the treatment of hemochromatosis,

comprising a nucleic acid for inhibiting expression of TMPRSS6 anddeferiprone, wherein the nucleic acid comprises at least one duplexregion that comprises at least a portion of a first strand and at leasta portion of a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of RNA transcribed from the TMPRSS6gene, wherein the first strand comprises the nucleotide sequence of SEQID NO:17 and the second strand comprises the nucleotide sequence of SEQID NO:18.

13. A nucleic acid for use as a medicament, preferably a nucleic acidfor use in the treatment of hemochromatosis, porphyria cutanea tarda,blood disorders, such as β-thalassemia or sickle cell disease,congenital dyserythropoietic anemia, marrow failure syndromes,myelodysplasia, transfusional iron overload, a disorder associated withiron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich'sAtaxia associated with iron overload, and infections and non-relapserelated mortality associated with bone marrow transplantation,preferably for the treatment of hemochromatosis,

wherein the nucleic acid inhibits expression of TMPRSS6, wherein thenucleic acid comprises at least one duplex region that comprises atleast a portion of a first strand and at least a portion of a secondstrand that is at least partially complementary to the first strand,wherein said first strand is at least partially complementary to atleast a portion of RNA transcribed from the TMPRSS6 gene, wherein thefirst strand comprises the nucleotide sequence of SEQ ID NO:17 and thesecond strand comprises the nucleotide sequence of SEQ ID NO:18, and

wherein the nucleic acid is administered with deferiprone.

14. The combination, combination for use, nucleic acid for use, kit,composition or composition for use according to any of the preferredembodiments 1-13, wherein the nucleic acid and one or more ironchelators are administered simultaneously, separately or sequentially.Preferably the nucleic acid and the chelator are administered not in asingle dose but separately. This allows to adapt the dosage of thenucleic acid and especially of the chelator to the need of the subject.

15. The combination, combination for use, nucleic acid for use, kit,composition or composition for use according to any of the preferredembodiments 1-14, wherein the nucleic acid is conjugated to a ligand,optionally at the 5′ end of the second strand, optionally wherein theligand comprises (i) one or more N-acetyl galactosamine (GalNAc)moieties and derivatives thereof, and (ii) a linker, wherein the linkerconjugates the GalNAc moieties to the nucleic acid.

16. The combination, combination for use, nucleic acid for use, kit,composition or composition for use according to preferred embodiment 15,wherein the conjugated nucleic acid has the structure:

wherein Z is a nucleic acid according to any of preferred embodiments 1to 14.

17. A combination, combination for use, or nucleic acid for useaccording to any one of preferred embodiments 1 to 14, or a conjugatednucleic acid according to preferred embodiments 15-16, wherein thenucleic acid is stabilised at the 5′ and/or 3′ end of either or bothstrands, optionally

wherein the nucleic acid comprises two phosphorothioate linkages betweeneach of the three terminal 3′ and between each of the three terminal 5′nucleotides on the first strand, and two phosphorothioate linkagesbetween the three terminal nucleotides of the 3′ end of the secondstrand, having the structure

5′-3′ TMPRSS6-hcm-9A 6 (ps) 2 (ps) 736462826472845 (ps) 4 (ps) 6 5′-3′TMPRSS6-hcm-9B 17273547153517184 (ps) 5 (ps) 1

wherein the specific modifications are depicted by the following numbers

1=2′F-dU,

2=2′F-dA,

3=2′F-dC,

4=2′F-dG,

5=2′-OMe-rU;

6=2′-OMe-rA;

7=2′-OMe-rC;

8=2′-OMe-rG

(ps)=phosphorothioate linkage.

18. A method of treating a disease or disorder, preferablyhemochromatosis, comprising administration of a combination comprising anucleic acid or conjugated nucleic acid and one or more iron chelatorsaccording to any of the preferred embodiments 1-17 to an individual inneed of treatment thereof.

The first strand of the nucleic acid preferably comprises, or consistsof the nucleotide sequence SEQ ID NO: 333, preferably as modified in SEQID NO: 17 and optionally as stabilised with phosphorothioate linkages asshown in SEQ ID NO: 217.

The second strand of the nucleic acid preferably comprises, or consistsof the nucleotide sequence SEQ ID NO: 334, preferably as modified in SEQID NO: 18 and optionally as stabilised with phosphorothioate linkages asshown in SEQ ID NO: 218. The ligand can be the ligand as shown in SEQ IDNO: 218 or a different ligand, but the ligand is preferably as shown inSEQ ID NO: 218.

The nucleic acid is preferably conjugated to a ligand, optionally at the5′ end of the second strand, optionally wherein the ligand is GN (FIG.8a ), GN2 (FIG. 8b ) or GN3 (FIG. 8c ), more preferably GN2.

The first stand of the nucleic acid preferably comprises, or consists ofSEQ ID NO: 217 and/or the second strand of the nucleic acid preferablycomprises, or consists of SEQ ID NO: 218, in both cases with allmodifications, including internucleotide modifications, and with theligand as shown.

Preferably, the disease or disorder to be treated is hemochromatosis,and most preferably hereditary hemochromatosis. Alternatively, or inaddition, the disease or disorder is preferably iron overload, such astransfusional iron overload, a disorder associated with iron overload orFriedreich's Ataxia associated with iron overload

Preferably, multiple doses of the siRNA are administered. The nucleicacid disclosed herein, or compositions including the same, may be foruse in a regimen comprising treatments once or twice weekly, every week,every two weeks, every three weeks, every four weeks, every five weeks,every six weeks, every seven weeks, or every eight weeks, or in regimenswith varying dosing frequency such as combinations of thebefore-mentioned intervals. In one embodiment, a subject is administeredan initial dose and one or more maintenance doses of a nucleic acidagent. The maintenance dose or doses can be the same or lower than theinitial dose, e.g., one-half less of the initial dose. The maintenancedoses are, for example, administered no more than once every 2, 5, 10,or 30 days.

The preferred embodiments can be combined with any other aspects,embodiments, statements and features disclosed herein.

Certain preferred features are listed below:

Statements

1. A nucleic acid for inhibiting expression of TMPRSS6, comprising atleast one duplex region that comprises at least a portion of a firststrand and at least a portion of a second strand that is at leastpartially complementary to the first strand, wherein said first strandis at least partially complementary to at least a portion of RNAtranscribed from the TMPRSS6 gene, wherein said first strand comprises anucleotide sequence selected from the following sequences: SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, or 215.

2. A nucleic acid of statement 1, wherein the second strand comprises anucleotide sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, or 216.

3. A nucleic acid of statement 1 or statement 2, wherein said firststrand comprises a nucleotide sequence of SEQ ID NO:17.

4. A nucleic acid of any one of statements 1 to 3, wherein said secondstrand comprises the nucleotide sequence of SEQ ID NO:18.

5. A nucleic acid according to any one of statements 1 to 4, whereinsaid first strand and/or said second strand are each from 17-35nucleotides in length.

6. A nucleic acid of any one of statements 1 to 5, wherein the at leastone duplex region consists of 19-25 consecutive nucleotide base pairs.

7. A nucleic acid of any preceding statement, which

-   -   a) is blunt ended at both ends; or    -   b) has an overhang at one end and a blunt end at the other; or    -   c) has an overhang at both ends.

8. A nucleic acid according to any preceding statement, wherein one ormore nucleotides on the first and/or second strand are modified, to formmodified nucleotides.

9. A nucleic acid of statement 8, wherein one or more of the oddnumbered nucleotides of the first strand are modified.

10. A nucleic acid according to statement 9, wherein one or more of theeven numbered nucleotides of the first strand are modified by at least asecond modification, wherein the at least second modification isdifferent from the modification of statement 9.

11. A nucleic acid of statement 10, wherein at least one of the one ormore modified even numbered nucleotides is adjacent to at least one ofthe one or more modified odd numbered nucleotides.

12. A nucleic acid of any one of statements 9 to 11, wherein a pluralityof odd numbered nucleotides are modified.

13. A nucleic acid of statement 10 to 12, wherein a plurality of evennumbered nucleotides are modified by a second modification.

14. A nucleic acid of any of statements 8 to 13, wherein the firststrand comprises adjacent nucleotides that are modified by a commonmodification.

15. A nucleic acid of any of statements 9 to 14, wherein the firststrand comprises adjacent nucleotides that are modified by a secondmodification that is different to the modification of statement 9.

16. A nucleic acid of any of statements 9 to 15, wherein one or more ofthe odd numbered nucleotides of the second strand are modified by amodification that is different to the modification of statement 9.

17. A nucleic acid according to any of statements 9 to 15, wherein oneor more of the even numbered nucleotides of the second strand aremodified by the modification of statement 9.

18. A nucleic acid of statement 16 or 17, wherein at least one of theone or more modified even numbered nucleotides of the second strand isadjacent to the one or more modified odd numbered nucleotides of thesecond strand.

19. A nucleic acid of any of statements 16 to 18, wherein a plurality ofodd numbered nucleotides of the second strand are modified by a commonmodification.

20. A nucleic acid of any of statements 16 to 19, wherein a plurality ofeven numbered nucleotides are modified by a modification according tostatement 9.

21. A nucleic acid of any of statements 16 to 20, wherein a plurality ofodd numbered nucleotides on the second strand are modified by a secondmodification, wherein the second modification is different from themodification of statement 9.

22. A nucleic acid of any of statements 16 to 21, wherein the secondstrand comprises adjacent nucleotides that are modified by a commonmodification.

23. A nucleic acid of any of statements 16 to 22, wherein the secondstrand comprises adjacent nucleotides that are modified by a secondmodification that is different from the modification of statement 9.

24. A nucleic acid according to any one of statements 8 to 23, whereineach of the odd numbered nucleotides in the first strand and each of theeven numbered nucleotides in the second strand are modified with acommon modification.

25. A nucleic acid of statement 24, wherein each of the even numberednucleotides are modified in the first strand with a second modificationand each of the odd numbered nucleotides are modified in the secondstrand with a second modification.

26. A nucleic acid according to any one of statements 8 to 25, whereinthe modified nucleotides of the first strand are shifted by at least onenucleotide relative to the unmodified or differently modifiednucleotides of the second strand.

27. A nucleic acid according to any one of statements 8 to 26, whereinthe modification and/or modifications are each and individually selectedfrom the group consisting of 3′-terminal deoxy-thymine, 2′-O-methyl, a2′-deoxy-modification, a 2′-amino-modification, a 2′-alkyl-modification,a morpholino modification, a phosphoramidate modification,5′-phosphorothioate group modification, a 5′ phosphate or 5′ phosphatemimic modification and a cholesteryl derivative or a dodecanoic acidbisdecylamide group modification.

28. A nucleic acid according to any one of statements 8 to 27, whereinthe modification is any one of a locked nucleotide, an abasic nucleotideor a non-natural base comprising nucleotide.

29. A nucleic acid according to any one of statements 8 to 28, whereinat least one modification is 2′-O-methyl.

30. A nucleic acid according to any one of statements 8 to 29, whereinat least one modification is 2′-F.

31. A nucleic acid for inhibiting expression of TMPRSS6 in a cell,comprising at least one duplex region that comprises at least a portionof a first strand and at least a portion of a second strand that is atleast partially complementary to the first strand, wherein said firststrand is at least partially complementary to at least a portion of aRNA transcribed from the TMPRSS6 gene, wherein said first strandcomprises a nucleotide sequence selected from the following sequences:SEQ ID Nos:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215,wherein the nucleotides of first strand are modified by firstmodification on the odd numbered nucleotides, and modified by a secondmodification on the even numbered nucleotides, and nucleotides of thesecond strand are modified by a third modification on the even numberednucleotides and modified by a fourth modification on the odd numberednucleotides, wherein at least the first modification is different to thesecond modification and the third modification is different to thefourth modification.

32. A nucleic acid of statement 31, wherein second sequence comprises anucleotide sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, or 216.

33. A nucleic acid of statement 31 or 32, wherein the fourthmodification and the second modification are the same.

34. A nucleic acid of any one of statements 31 to 33, wherein the firstmodification and the third modification are the same.

35. A nucleic acid of any one of statements 31 to 34, wherein the firstmodification is 2′O-Me and the second modification is 2′F.

36. A nucleic acid of any one of statements 31 to 35, wherein the firststrand comprises the nucleotide sequence of SEQ ID NO:17 and the secondstrand comprises the nucleotide sequence of SEQ ID NO:18.

37. A nucleic acid of any one of statements 31 to 36, comprising asequence and modifications as shown in the table below:

SEQ ID 5′aaccagaagaagca 6273646282647284546 NO: 17 gguga 3′ SEQ ID5′ucaccugcuucuuc 1727354715351718451 NO: 18 ugguu 3′

wherein, the specific modifications are depicted by numbers

1=2′F-dU,

2=2′F-dA,

3=2′F-dC,

4=2′F-dG,

5=2′-OMe-rU;

6=2′-OMe-rA;

7=2′-OMe-rC;

8=2′-OMe-rG.

38. A nucleic acid according to any one of statements 1 to 37,conjugated to a ligand.

39. A nucleic acid according to any one of statements 1 to 38,comprising a phosphorothioate linkage between the terminal one, two orthree 3′ nucleotides and/or 5′ nucleotides of the first and/or thesecond strand.

40. A nucleic acid according to any one of statements 1 to 39 comprisingtwo phosphorothioate linkage between each of the three terminal 3′ andbetween each of the three terminal 5′ nucleotides on the first strand,and two phosphorothioate linkages between the three terminal nucleotidesof the 3′ end of the second strand.

41. A nucleic acid for inhibiting expression of TMPRSS6 in a cell,comprising at least one duplex region that comprises at least a portionof a first strand and at least a portion of a second strand that is atleast partially complementary to the first strand, wherein said firststrand is at least partially complementary to at least a portion of aRNA transcribed from the TMPRSS6 gene, wherein said first strandcomprises a nucleotide sequence selected from the following sequences:SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215,wherein said nucleic acid conjugated to a ligand.

42. A nucleic acid according to statement 41, wherein the ligandcomprises (i) one or more N-acetyl galactosamine (GalNAc) moieties andderivatives thereof, and (ii) a linker, wherein the linker conjugatesthe GalNAc moieties to a nucleic acid as defined in statement 41

43. A nucleic acid according to statement 41 or 42, wherein linker maybe a bivalent or trivalent or tetravalent branched structure.

44. A nucleic acid according to any of statements 41 to 43, wherein thenucleotides are modified as defined in any preceding statements.

45. A nucleic acid of any preceding statement, wherein the ligandcomprises the formula I:

[S-X¹-P-X²]₃-A-linker-  (I)

wherein:

-   -   S represents a saccharide, wherein the saccharide is N-acetyl        galactosamine;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is alkylene or an alkylene ether of the formula        (—CH₂)_(n)—O—CH₂— where n=1-6;    -   A is a branching unit;    -   X³ represents a bridging unit;    -   wherein a nucleic acid as defined in any of statements 1 to 40        is conjugated to X³ via a phosphate or modified phosphate        (preferably a thiophosphate).

46. A conjugated nucleic acid having one of the following structures

wherein Z is a nucleic acid according to any of statements 1 to 40.

47. A nucleic acid according to any of statements 1 to 40, which isconjugated to a ligand of the following structure

48. A nucleic acid or conjugated nucleic acid of any precedingstatement, wherein the duplex comprises separate strands.

49. A nucleic acid or conjugated nucleic acid of any precedingstatement, wherein the duplex comprises a single strand comprising afirst strand and a second strand.

50. A composition comprising a nucleic acid or conjugated nucleic acidas defined in any preceding statement and a formulation comprising:

-   -   i) a cationic lipid, or a pharmaceutically acceptable salt        thereof;    -   ii) a steroid;    -   iii) a phosphatidylethanolamine phospholipid;    -   iv) a PEGylated lipid.

51. A composition according to statement 50, wherein in the formulationthe content of the cationic lipid component is from about 55 mol % toabout 65 mol % of the overall lipid content of the lipid formulation,preferably about 59 mol % of the overall lipid content of the lipidformulation.

52. A composition as statemented in statement 50, wherein theformulation comprises; A cationic lipid having the structure;

the steroid has the structure;

the a phosphatidylethanolamine phospholipid has the structure;

And the PEGylated lipid has the structure;

53. A composition comprising a nucleic acid or conjugated nucleic acidof any preceding statement and a physiologically acceptable excipient.

54. A nucleic acid or conjugated nucleic acid according to any precedingstatement for use in the treatment of a disease or disorder.

55. Use of a nucleic acid or conjugated nucleic acid according to anypreceding statement in the manufacture of a medicament for treating adisease or disorder.

56. A method of treating a disease or disorder comprising administrationof a composition comprising a nucleic acid or conjugated nucleic acidaccording to any one preceding statement to an individual in need oftreatment.

57. The method of statement 55, wherein the nucleic acid or conjugatednucleic acid is administered to the subject subcutaneously orintravenously.

58. Use or method according to any of statements 54 to 56, wherein saiddisease or disorder is selected from the group comprisinghemochromatosis, erythropoietic porphyria, transfusional iron overloadand blood disorders.

59. Use or method according to statement 58, wherein the blood disorderis β-thalassemias, sickle cell anaemia, congenital sideroblastic anemia,aplastic anemia or myelodysplastic syndrome.

60. Use or method according to any of statements 54 to 59, wherein thedisorder is associated with iron overload.

61. Use or method according to statement 60, wherein the disorderassociated with iron overload is Parkinson's Disease, Alzheimer'sDisease or Friedreich's Ataxia.

62. A process for making a nucleic acid or conjugated nucleic acidaccording to any of statements 1 to 49.

The invention will now be described with reference to the followingnon-limiting figures and examples in which:

FIG. 1 shows the results of an RNAi molecule screen for inhibition ofTMPRSS6 expression in human Hep3B cells;

FIG. 2 shows the dose response of TMPRSS6 RNAi molecules in human Hep3Bcells;

FIG. 3 shows the reduction of TMPRSS6 expression in mouse liver tissueby different doses of GalNAc siRNA molecules;

FIG. 4 shows the duration of TMPRSS6 target gene inhibition by TMPRSS6siRNA molecules and the induction of HAMP mRNA expression in mice;

FIG. 5 shows that the inhibition of TMPRSS6 expression by treatment withTMPRSS6 siRNA molecules reduces iron levels in serum for an extendedtime;

FIG. 6 shows the reduction of TMRPSS6 expression in 1° mouse hepatocytesby receptor mediated uptake of GalNAc conjugated RNAi molecules;

FIG. 7 shows the GalNAc conjugated RNAi molecules used in examples 1, 3,4, 26 to 31 and 34 to 38;

FIGS. 8a, 8b and 8c show the structure of the GalNAc ligands referred toherein respectively as GN, GN2, and GN3 to which the oligonucleotideswere conjugated (see also below this nomenclature in the Examples whereTMPRSS6 hcm-9 is conjugated to each of GN, GN2 and GN3);

FIG. 9 shows the reduction of TMPRSS6 expression by different siRNAmodification variants in human Hep3B cells;

FIG. 10 shows the reduction of TMPRSS6 expression by differentGalNAc-siRNA conjugates by receptor mediated uptake;

FIG. 11 shows the reduction of TMPRSS6 and the induction of HAMPexpression by different GalNAc-siRNA conjugates in mice;

FIG. 12 shows the sequences and modifications of the GalNAc-siRNAmolecules of Examples 8, 9 and 10;

FIG. 13 shows the reduction of TMPRSS6 expression in the liver tissueand the reduction of serum iron levels in mice at different time pointsafter single injection with siRNA conjugates;

FIG. 14 shows the reduction of TMPRSS6 mRNA levels in liver and thereduction of serum iron levels in mice by different doses of GalNAcconjugated siRNA molecules;

FIG. 15 shows the effect of different modification patterns on theactivity of an siRNA molecule in human Hep3B cells;

FIG. 16 shows the effect of different modification variants of GalNAcconjugated siRNA molecules on inhibition of TMPRSS6 expression in humanHep3B cells;

FIG. 17 shows the effect of a modification variant of a GalNAcconjugated siRNAs on inhibition of TMPRSS6 expression in primary mousehepatocytes by receptor mediated uptake;

FIG. 18 shows the reduction of TMPRSS6 expression by different siRNAmodification variants in human Hep3B cells;

FIG. 19 shows the influence of inverted A and G nucleotides on RNAiactivity in human Hep3B cells;

FIG. 20 shows the influence of inverted RNA nucleotides on siRNAstability;

FIG. 21 shows the influence of inverted RNA nucleotides on siRNAactivity in human Hep3B cells;

FIG. 22 shows the influence of inverted RNA nucleotides on RNAi activityin human Hep3B cells;

FIG. 23 shows the influence of inverted RNA nucleotides on the activityof GalNAc conjugated siRNAs in primary mouse hepatocytes by receptormediated uptake;

FIG. 24 shows the effect of phosphorodithioate linkage on stability ofGalNAc conjugated siRNA molecules;

FIG. 25 shows the effect of phosphorodithioate linkage on activity of aGalNAc conjugated siRNA molecule on TMPRSS6 expression in mouse primaryhepatocytes;

FIG. 26 shows the effect of phosphorodithioate linkage on stability of aGalNAc conjugated siRNA molecule;

FIG. 27 shows the inhibition of TMPRSS6 expression in mouse primaryhepatocytes by a GalNAc siRNA conjugate containing phosphorodithioatelinkages;

FIG. 28 shows the reduction of TMPRSS6 expression by different doses ofGalNAc siRNAs in an animal model for hereditary hemochromatosis;

FIG. 29 shows the increase of serum Hepcidin levels by different dosesof GalNAc siRNAs in an animal model for hereditary hemochromatosis;

FIG. 30 shows the reduction of serum iron levels by different doses ofGalNAc siRNAs in an animal model for hereditary hemochromatosis;

FIG. 31 shows the reduction of transferrin saturation by different dosesof GalNAc siRNAs in an animal model for hereditary hemochromatosis;

FIG. 32 shows the increase in Unsaturated Iron Binding Capacity bydifferent doses of GalNAc siRNAs in an animal model for hereditaryhemochromatosis;

FIG. 33 shows the reduction of tissue iron levels by GalNAc siRNAs in ananimal model for hereditary hemochromatosis;

FIG. 34 shows the reduction of human TMPRSS6 mRNA levels in Hep3B cellsby liposomal delivery of 43 additional siRNAs;

FIG. 35 shows the dose response curves of different siRNAs forinhibition of TMPRSS6 expression in Hep3B cells;

FIG. 36 shows the reduction of TMPRSS6 expression in primary humanhepatocytes by receptor mediated uptake of GalNAc siRNA conjugates atdifferent concentrations;

FIG. 37 shows the increase of haematocrit values in rodent model forβ-thalassemia intermedia by treatment with GalNAc siRNA conjugates;

FIG. 38 shows the reduction in red blood cell distribution widths inrodent model for β-thalassemia intermedia by treatment with GalNAc siRNAconjugates.

FIG. 39 shows the reduction in proportion of reticulocytes in blood ofrodent model for β-thalassemia intermedia by treatment with GalNAc siRNAconjugates;

FIG. 40 shows the reduction of reactive oxygen species in red bloodcells of rodent model for β-thalassemia intermedia by treatment withGalNAc siRNA conjugates.

FIG. 41 shows GalNAc TMPRSS6 siRNA raises hemoglobin levels in rodentmodel for β-thalassemia intermedia.

FIG. 42 shows GalNAc TMPRSS6 reduces splenomegaly in rodent model forβ-thalassemia intermedia.

FIG. 43 shows GalNAc TMPRSS6 improves red blood cell maturation in thebone marrow.

FIG. 44 shows GalNAc TMPRSS6 reduces ineffective erythropoiesis in thespleen.

FIG. 45 a and b show dose-response curves of siRNA conjugates againstTMPRSS6 in primary human hepatocytes.

FIG. 46 shows inhibition of TMPRSS6 mRNA expression by siRNA conjugatesin primary human hepatocytes.

FIG. 47 shows sequences and modification pattern of GalNAc siRNAconjugates that were tested for inhibition of TMPRSS6 expression inprimary human hepatocytes.

FIG. 48 shows specific sequences of nucleic acids used in Example 44.

FIG. 49 shows receptor-mediated uptake in primary mouse hepatocytes byGalNAc siRNA conjugates targeting TMPRSS6 containing different endstabilization chemistries (phosphorothioate, phosphorodithioate,phosphodiester).

FIG. 50 shows serum stability of GalNAc-siRNA conjugates withphosphorothioates, phosphorodithioates and phosphodiesters in terminalpositions and in the GalNAc moiety FIG. 51 shows inhibition of TMPRSS6gene expression in primary murine hepatocytes 24 h following treatmentwith TMPRSS6-siRNA carrying vinyl-(E)-phosphonate 2′OMe-Uracil at the5′-position of the anti-sense strand and two phosphorothioate linkagesbetween the first three nucleotides (X0204), vinyl-(E)-phosphonate2′OMe-Uracil at the 5′-position of the anti-sense strand andphosphodiester bonds between the first three nucleotides (X0205),(X0139) or tetrameric (X0140)) or a tree like trimeric GalNAc-cluster(X0004) or a non-targeting GalNAc-siRNA (X0028) at indicatedconcentrations or left untreated (UT).

FIG. 52 shows serum stability of siRNA-conjugates vs. less stabilizedpositive control for nuclease degradation.

FIG. 53 shows reduction of transferrin saturation by TMPRSS6 siRNAtreatment in animal model for hereditary hemochromatosis type 1.

FIG. 54 shows treatment with TMPRSS6 siRNAs enhances the reduction oftissue iron levels by iron chelator.

EXAMPLES Example 1

Nucleic acids in accordance with the disclosure were synthesised, usingthe oligos as set out in the tables below.

The method of synthesis was as follows, using one of the sequences ofthe disclosure as an example:

STS012 (GN-TMPRSS6-hcm-9)

First strand

5′mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG(ps) mA 3′

Second strand

5′[ST23 (ps)]3 long trebler (ps) fU mC fA mC fC mU fG mC fU mU fC mU fUmC fU mG fG (ps) mU (ps) fU 3′

fN (N=A, C, G, U) denotes 2′Fluoro, 2′ DeoxyNucleosides

mN (N=A, C, G, U) denotes 2′O Methyl Nucleosides

(ps) indicates a phosphorothioate linkage

ST23 is a GalNAc C4 phosphoramidite (structure components as below)

Long trebler (STKS)

A further example is GN2-TMPRSS6-hcm-9 (STS12009L4):

First strand

5′mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG(ps) mA 3′

Second strand

5′[ST23 (ps)]3 ST41 (ps) fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mGfG (ps) mU

(ps) fU 3′

fN (N=A, C, G, U) denotes 2′Fluoro, 2′ DeoxyNucleosides

mN (N=A, C, G, U) denotes 2′O Methyl Nucleosides

(ps) indicates a phosphorothioate linkage

ST23 is as above.

ST41 is as follows (and as described in WO2017/174657):

All Oligonucleotides were either obtained from a commercialoligonucleotide manufacturer (Eurogentech, Belgium; Biospring, Germany)or synthesized on an AKTA oligopilot synthesizer using standardphosphoramidite chemistry. Commercially available solid support and2′O-Methyl RNA phosphoramidtes, 2′Fluoro DNA phosphoramidites (allstandard protection) and commercially available long treblerphosphoramidite (Glen research) were used. Synthesis was performed using0.1 M solutions of the phosphoramidite in dry acetonitrile andbenzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile).All other reagents were commercially available standard reagents.

Synthesis of PS2 containing oligonucleotides was performed according tothe instructions of the manufacturer (Glen Research, AM Biotech).Vinyl-(E)-phosphonate 2′OMe-Uracil phosphoramidite was synthesized andused in oligonucleotide synthesis according to literature publishedmethods (Haraszti et al., Nuc. Acids Res., 45(13), 2017, 7581-7592).

Conjugation of the GalNAc synthon (ST23) was achieved by coupling of therespective phosphoramidite to the 5′end of the oligochain under standardphosphoramidite coupling conditions. Phosphorothioates were introducedusing standard commercially available thiolation reagents (EDITH, Linktechnologies).

The single strands were cleaved off the CPG by using Methylamine. WhereTBDMS protected RNA nucleosides were used, additional treatment withTEA*3HF was performed to remove the silyl protection. The resultingcrude oligonucleotide was purified by ion exchange chromatography(Resource Q, 6 mL, GE Healthcare) on a AKTA Pure HPLC System using aSodium chloride gradient. Product containing fractions were pooled,desalted on a size exclusion column (Zetadex, EP Biotech) andlyophilised.

For annealing, equimolar amounts of the respective single strands weredissolved in water and heated to 80° C. for 5 min. After cooling theresulting Duplex was lyophilised.

The sequences of the resulting nucleic acids (siNAs) are set out inTable 1.

TABLE 1nucleic acid sequences tested for inhibition of TMPRSS6 expression.Nucleic acids were synthesized by Biospring, Frankfurt.Nucleotides modifications are depicted by the following numbers(column 4), 1 = 2′F-dU, 2 = F′-dA, 3 = 2F′-dC, 4 = 2′F-dG,5 = 2′-OMe-dU; 6 = 2′-OMe-rA; 7 = 2′-OMe-dC; 8 = 2′-OMe-dG. SEQ IDName - NO: TMPRSS6- . . . Sequence Modifications 1 hc-1A5′augucuuucacacuggcuu 3′ 6181715172727184715 2 hc-1B5′aagccagugugaaagacau 3′ 2647364545462646361 3 h-2A5′auugaguacacgcagacug 3′ 6154645272747282718 4 h-2B5′cagucugcguguacucaau 3′ 3645354745452717261 5 h-3A5′aaguugauggugaucccgg 3′ 6281546184546173748 6 h-3B5′ccgggaucaccaucaacuu 3′ 3748461727361726351 7 hc-4A5′uucuggaucguccacuggc 3′ 5171846174537271847 8 hc-4B5′gccaguggacgauccagaa 3′ 4736454827461736462 9 h-5A5′auucacagaacagaggaac 3′ 6153636462728284627 10 h-5B5′guuccucuguucugugaau 3′ 4517353545171818261 11 h-6A5′guagucauggcuguccucu 3′ 8164536184718173535 12 h-6B5′agaggacagccaugacuac 3′ 2828463647361827163 13 h-7A5′aguuguaguaaguucccag 3′ 6451816452645173728 14 h-7B5′cugggaacuuacuacaacu 3′ 3548462715271636271 15 hcmr-8A5′uuguacccuaggaaauacc 3′ 5181637352846261637 16 hcmr-8B5′gguauuuccuaggguacaa 3′ 4816151735284816362 17 hcm-9A5′aaccagaagaagcagguga 3′ 6273646282647284546 18 hcm-9B5′ucaccugcuucuucugguu 3′ 1727354715351718451 19 hc-10A5′uaacaacccagcguggaau 3′ 5263627372838184625 20 hc-10B5′auuccacgcuggguuguua 3′ 2517363835484518152 21 hc-11A5′guuucucucauccaggccg 3′ 8151717172537284738 22 hc-11B5′cggccuggaugagagaaac 3′ 3847354825464646263 23 hcm-12A5′gcaucuucugggcuuuggc 3′ 8361715354847151847 24 hcm-12B5′gccaaagcccagaagaugc 3′ 4736264737282646183 25 hc-13A5′ucacacuggaaggugaaug 3′ 5363635482648182618 26 hc-13B5′cauucaccuuccaguguga 3′ 3615363715372818182 27 hcmr-14A5′cacagaugugucgaccccg 3′ 7272825454538273738 28 hcmr-14B5′cggggucgacacaucugug 3′ 3848453827272535454 29 hcmr-15A5′uguacccuaggaaauacca 3′ 5452737164826252736 30 hcmr-15B5′ugguauuuccuaggguaca 3′ 1845251537164845272 31 Luc-siRNA-1A5′ucgaaguauuccgcguacg 3′ 5382645251738381638 32 Luc-siRNA-1B5′cguacgcggaauacuucga 3′ 3816383856252715382 33 PTEN-A5′uaaguucuagcuguggugg 3′ 5a6g5u7u6g7u8u8g5g8 34 PTEN-B5′ccaccacagcuagaacuua 3′ c7a7c6c6g7u6g6a7u5a

TABLE 2 the start position of each nucleic acid sequencewithin the TMPRSS6 mRNA sequence Ref NM_001289000.1. SEQ CorrespondingID Start Oligo nucleic acid NO: 253 GGUAUUUCCUAGGGUACAA TMPRSS6-hcmr-816 305 CAGUCUGCGUGUACUCAAU TMPRSS6-h-2 4 381 GCCAAAGCCCAGAAGAUGCTMPRSS6-hcm-12 24 426 CUGGGAACUUACUACAACU TMPRSS6-h-7 14 478UCACCUGCUUCUUCUGGUU TMPRSS6-hcm-9 18 652 AAGCCAGUGUGAAAGACAUTMPRSS6-hc-1 2 682 AUUCCACGCUGGGUUGUUA TMPRSS6-hc-10 20 1234GCCAGUGGACGAUCCAGAA TMPRSS6-hc-4 8 1318 CCGGGAUCACCAUCAACUU TMPRSS6-h-36 1418 GUUCCUCUGUUCUGUGAAU TMPRSS6-h-5 10 1481 CGGCCUGGAUGAGAGAAACTMPRSS6-hc-11 22 1633 CAUUCACCUUCCAGUGUGA TMPRSS6-hc-13 26 1824CGGGGUCGACACAUCUGUG TMPRSS6-hcmr-14 28 2018 AGAGGACAGCCAUGACUACTMPRSS6-h-6 12 252 UGGUAUUUCCUAGGGUACA TMPRRS6-hcmr-15 30

Screen for Inhibition TMPRSS6 Expression in Human Hep3B Cells.

Cells were plated at a cell density of 80,000 cells per 6 well dish. Thefollowing day cells were transfected with 20 nM nucleic acid (listed inTable 1) and 1 μg/ml AtuFECT (50:50 formulation of cationic lipidAtufect01 and fusogenic lipid DPhyPE.). Two days after transfectioncells were lysed and TMPRSS6 mRNA levels were determined by q-RT-PCRusing the amplicons in Table 3. TMPRSS6 mRNA levels were normalized toexpression levels of the house keeping gene PTEN. Nucleic acids forLuciferase and PTEN were used as non targeting control nucleic acids.Results are shown in FIG. 1.

TABLE 3 Sequences of TMPRSS6, Actin, PTEN and HAMP amplicon setsthat were used to measure mRNA levels of respective genes. SEQ ID NO:hTMPRSS6 (upper) 5′CCGCCAAAGCCCAGAAG 3′ 35 hTMPRSS6 (lower)5′GGTCCCTCCCCAAAGGAATAG 3′ 36 hTMPRSS6 (probe)5′CAGCACCCGCCTGGGAACTTACTACAAC 3′ 37 mTMPRSS6 (upper)5′CGGCACCTACCTTCCACTCTT 3′ 38 mTMPRSS6 (lower) 5′TCGGTGGTGGGCATCCT 3′ 39mTMPRSS6 (probe) 5′CCGAGATGTTTCCAGCTCCCCTGTTCTA 3′ 40 h-Aktin (upper)5′GCATGGGTCAGAAGGATTCCTAT 3′ 41 h-Aktin (lower)5′TGTAGAAGGTGTGGTGCCAGATT 3′ 42 h-Aktin (probe)5′TCGAGCACGGCATCGTCACCAA 3′ 43 mAktin (upper)5′GTTTGAGACCTTCAACACCCCA 3′ 44 mAktin (lower) 5′GACCAGAGGCATACAGGGACA 3′45 mAktin (probe) 5′CCATGTACGTAGCCATCCAGGCTGTG 3′ 46 PTEN (upper)5′CACCGCCAAATTTAACTGCAGA 3′ 47 PTEN (lower)5′AAGGGTTTGATAAGTTCTAGCTGT 3′ 48 PTEN (probe)5′TGCACAGTATCCTTTTGAAGACCATAACCCA 3′ 49 mHAMP: (upper)5′CCTGTCTCCTGCTTCTCCTCCT 3′ 50 mHAMP: (lower)5′AATGTCTGCCCTGCTTTCTTCC 3′ 51 mHAMP: (probe)5′TGAGCAGCACCACCTATCTCCATCAACA3′ 52

Example 2

Dose Response of TMPRSS6 Nucleic Acids for Inhibition of TMPRSS6Expression in Human Hep3B Cells.

Cells were plated at a cell density of 150,000 cells per 6 well dish.The following day cells were transfected with 1 μg/ml AtuFECT (50:50formulation of cationic lipid Atufect01 and fusogenic lipid DPhyPE.) anddifferent amounts of TMPRSS6 nucleic acids (30; 10; 3; 1; 0.3; 0.1,0.003 nM, respectively) as depicted by the X-axis of the graph in FIG.2. For non targeting control samples, cells were transfected with 30 and10 nM nucleic acid for Luciferase. Two days after the transfection cellswere lysed and mRNA levels were determined by q-RT-PCR. TMPRSS6 mRNAlevels were normalized to the expression levels of the house keepinggene PTEN. The highest reduction of TMPRSS6 mRNA expression was observedby TMPRSS6-hcm9 nucleic acid. Transfection with Luciferase nucleic aciddid not affect TMPRSS6 mRNA levels. Results are shown in FIG. 2.Sequences of RNAi molecules are depicted in Table 1.

Example 3

Inhibition of TMPRSS6 Expression in Liver Tissue by Different Doses ofGalNAc Nucleic Acids.

C57/BL6 mice were treated with a single dose of 10, 3 or 1 mg/kg ofGalNAc nucleic acid conjugates by subcutaneous administration. Sequenceand modifications of respective siRNA conjugates are shown in FIG. 7.The siRNAs are conjugated to GalNAc linker (GN) depicted in FIG. 8a anddescribed therein. Control groups were treated with isotonic saline orwith non targeting control conjugate, GN-TTR-hc, respectively. Targetgene expression in liver tissue was assessed by qRT PCR three days aftersubcutaneous injection of the conjugates. Total RNA was isolated fromsnap frozen tissue samples and qRT-PCR was performed as describedpreviously (Kuhla et al. 2015, Apoptosis Vol 4, 500-11). TaqMan probesthat were used are shown in Table 3. Results are shown in FIG. 3.

Example 4

Duration of Target Gene Inhibition by TMPRSS6 RNAi Molecules.

Mice were treated with 3 mg/kg GalNAc-TMPRSS6 RNAi molecules bysubcutaneous injection. Sequence and modifications of respective siRNAconjugates are shown in FIG. 7. Target mRNA expression was assessed inliver tissue at day 7, 14, 21, 27, 34 or day 41 after treatment (dayspost injection, dpi). Reduction of TMPRSS6 expression in the liver isobserved until day 41 after injection. HAMP (hepcidin) mRNA expressionis upregulated in the liver of mice treated with GN-TMPRSS6 RNAimolecule. Results are shown in FIG. 4.

Example 5

Inhibition of TMPRSS6 Expression by Treatment with TMPRSS6 siRNA ReducesIron Levels in Blood.

Iron levels were analyzed in serum 7, 14, 21, 27, 34 and 41 days aftermice were treated subcutaneously with 3 mg/kg GalNAc nucleic acidconjugates. Serum iron levels were reduced up to 41 days post injection(dpi 41). Sequence and modifications of respective siRNA conjugates areshown in FIG. 7. Results are shown in FIG. 5.

Example 6

Inhibition of TMPRSS6 Expression by Receptor Mediated Uptake.

Primary mouse hepatocytes were plated on collagen coated dishes andincubated with siRNA conjugates diluted in cell culture medium at aconcentration of 100 nM to 0.03 nM as indicated. 24 hours after exposingthe cells to siRNA conjugates, total RNA was extracted and TMPRSS6expression was quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels werenormalized to Actin mRNA levels. Dose dependent inhibition of TMPRSS6expression was observed by both GalNAc-TMPRSS6 siRNA conjugates.GN2-Luc-siRNA1 GalNAc conjugate (GN2-Luc) was used as non targetingcontrol and did not affect TMPRSS6 mRNA expression. Sequence andmodifications of respective siRNA conjugates are depicted in FIG. 7.Results are shown in FIG. 6.

Example 7

Further TMPRSS6 siRNAs were synthesised, in accordance with the methoddescribed above. The sequences and modifications are shown in Table 4below:

Modification variants of an siRNA targeting TMPRSS6 (Sequence ID 17 andID18). For each duplex, the first sequence (A strand) is listed on topand the second sequence (B strand) below. All sequences correspond toSEQ ID NO:17 (top) and SEQ ID NO:18 (bottom). Modification codes arelisted at the end of the Table 4.

TABLE 4 Modifications are depicted by numbers shown in the rowsat the bottome of the table, and for each duplex the firststrand is on top and the second strand is below. Duplex A strandsequence sequence and chemistry ID B strand (5′-3′) (5′-3′) TMP01TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH01Bucaccugcuucuucugguu 1727354715351718451 TMP02 TMPJH02Aaaccagaagaagcagguga 2237242282243284142 TMPJH02B ucaccugcuucuucugguu1723314715355754815 TMP03 TMPJH03A aaccagaagaagcagguga2273282646283248182 TMPJH03B ucaccugcuucuucugguu 5363718351715354815TMP04 TMPJH04A aaccagaagaagcagguga 2273282646683248182 TMPJH03Bucaccugcuucuucugguu 5363718351715354815 TMP05 TMPJH01Aaaccagaagaagcagguga 6273646282647284546 TMPJH03B ucaccugcuucuucugguu5363718351715354815 TMP06 TMPJH05A aaccagaagaagcagguga2273242242643244542 TMPJH05B ucaccugcuucuucugguu 5727354315355754455TMP07 TMPJH06A aaccagaagaagcagguga 2277646242643244542 TMPJH05Bucaccugcuucuucugguu 5727354315355754455 TMP08 TMPJH07Aaaccagaagaagcagguga 2277686242643244542 TMPJH05B ucaccugcuucuucugguu5727354315355754455 TMP09 TMPJH08A aaccagaagaagcagguga2273242246687244542 TMPJH05B ucaccugcuucuucugguu 5727354315355754455TMP10 TMPJH09A aaccagaagaagcagguga 2273242682687244542 TMPJH05Bucaccugcuucuucugguu 5727354315355754455 TMP11 TMPJH10Aaaccagaagaagcagguga 2273242242643284586 TMPJH05B ucaccugcuucuucugguu5727354315355754455 TMP12 TMPJH11A aaccagaagaagcagguga2273242242643288586 TMPJH05B ucaccugcuucuucugguu 5727354315355754455TMP13 TMPJH12A aaccagaagaagcagguga 2273242246687284586 TMPJH05Bucaccugcuucuucugguu 5727354315355754455 TMP14 TMPJH13Aaaccagaagaagcagguga 6277646246687284586 TMPJH13B ucaccugcuucuucugguu5767354315755758855 TMP15 TMPJH14A aaccagaagaagcagguga2273282282283284182 TMPJH14B ucaccugcuucuucugguu 5327318315315318415TMP16 TMPJH15A aaccagaagaagcagguga 2273282282643284182 TMPJH14Bucaccugcuucuucugguu 5327318315315318415 TMP17 TMPJH16Aaaccagaagaagcagguga 2237242242247244582 TMPJH16B ucaccugcuucuucugguu1723314355311358411 TMP18 TMPJH10A aaccagaagaagcagguga2273242242643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451TMP19 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH02Bucaccugcuucuucugguu 1723314715355754815 TMP20 TMPJH10Aaaccagaagaagcagguga 2273242242643284586 TMPJH03B ucaccugcuucuucugguu5363718351715354815 TMP21 TMPJH10A aaccagaagaagcagguga2273242242643284586 TMPJH13B ucaccugcuucuucugguu 5767354315755758855TMP22 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH14Bucaccugcuucuucugguu 5327318315315318415 TMP23 TMPJH10Aaaccagaagaagcagguga 2273242242643284586 TMPJH16B ucaccugcuucuucugguu1723314355311358411 TMP24 TMPJH02A aaccagaagaagcagguga2237242282243284142 TMPJH01B ucaccugcuucuucugguu 1727354715351718451TMP25 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH03Bucaccugcuucuucugguu 5363718351715354815 TMP26 TMPJH02Aaaccagaagaagcagguga 2237242282243284142 TMPJH05B ucaccugcuucuucugguu5727354315355754455 TMP27 TMPJH02A aaccagaagaagcagguga2237242282243284142 TMPJH13B ucaccugcuucuucugguu 5767354315755758855TMP28 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH14Bucaccugcuucuucugguu 5327318315315318415 TMP29 TMPJH02Aaaccagaagaagcagguga 2237242282243284142 TMPJH16B ucaccugcuucuucugguu1723314355311358411 TMP30 TMPJH13A aaccagaagaagcagguga6277646246687284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451TMP31 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH02Bucaccugcuucuucugguu 1723314715355754815 TMP32 TMPJH13Aaaccagaagaagcagguga 6277646246687284586 TMPJH03B ucaccugcuucuucugguu5363718351715354815 TMP33 TMPJH13A aaccagaagaagcagguga6277646246687284586 TMPJH05B ucaccugcuucuucugguu 5727354315355754455TMP34 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH14Bucaccugcuucuucugguu 5327318315315318415 TMP35 TMPJH13Aaaccagaagaagcagguga 6277646246687284586 TMPJH16B ucaccugcuucuucugguu1723314355311358411 TMP36 TMPJH10A aaccagaagaagcagguga2273242242643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451TMP37 TMPJH17A aaccagaagaagcagguga 2273282242643284586 TMPJH01Bucaccugcuucuucugguu 1727354715351718451 TMP38 TMPJH18Aaaccagaagaagcagguga 6277682242643288142 TMPJH01B ucaccugcuucuucugguu1727354715351718451 TMP39 TMPJH19A aaccagaagaagcagguga6277682242643288586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451TMP40 TMPJH20A aaccagaagaagcagguga 2233282242643284142 TMPJH01Bucaccugcuucuucugguu 1727354715351718451 TMP41 TMPJH21Aaaccagaagaagcagguga 2277282242643284586 TMPJH01B ucaccugcuucuucugguu1727354715351718451 TMP42 TMPJH22A aaccagaagaagcagguga2273282642643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451TMP43 TMPJH23A aaccagaagaagcagguga 2273282282643284586 TMPJH01Bucaccugcuucuucugguu 1727354715351718451 TMP44 TMPJH24Aaaccagaagaagcagguga 2273282246643284586 TMPJH01B ucaccugcuucuucugguu1727354715351718451 TMP45 TMPJH25A aaccagaagaagcagguga2273282242683284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451TMP46 TMPJH26A aaccagaagaagcagguga 2273282242647284586 TMPJH01Bucaccugcuucuucugguu 1727354715351718451 TMP47 TMPJH01Aaaccagaagaagcagguga 6273646282647284546 TMPJH05B ucaccugcuucuucugguu5727354315355754455 TMP48 TMPJH01A aaccagaagaagcagguga6273646282647284546 TMPJH27B ucaccugcuucuucugguu 5727754715355754855TMP49 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH28Bucaccugcuucuucugguu 1723314311351714411 TMP50 TMPJH01Aaaccagaagaagcagguga 6273646282647284546 TMPJH29B ucaccugcuucuucugguu5767354315355754455 TMP51 TMPJH01A aaccagaagaagcagguga6273646282647284546 TMPJH30B ucaccugcuucuucugguu 5727754315355754455TMP52 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH31Bucaccugcuucuucugguu 5727358315355754455 TMP53 TMPJH01Aaaccagaagaagcagguga 6273646282647284546 TMPJH32B ucaccugcuucuucugguu5727354315755754455 TMP54 TMPJH01A aaccagaagaagcagguga6273646282647284546 TMPJH33B ucaccugcuucuucugguu 5727354315355758455 1= 2′F-dU 2 = 2′F-dA 3 = 2′F-dC 4 = 2′F-dG 5 = 2′OMe-rU 6 = 2′OMe-rA 7= 2′OMe-rC 8 = 2′OMe-rG

Different modification variants of one siRNA targeting TMPRSS6 weretested in human Hep3B cells. siRNAs targeting PTEN and Luciferase wereused as non-related and non-targeting controls, respectively. All siRNAswere transfected with 1 μg/ml Atufect at 1 nM (0.1 nM when indicated).Total RNA was extracted 48 h after transfection and TMPRSS6 mRNA levelswere quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalizedto Actin mRNA levels. Each bar represents mean+/−SD of three technicalreplicates. The results are shown in FIG. 9.

Example 8

Modification variants of a GalNAc-conjugated siRNA targeting TMPRSS6were synthesised and are shown in FIG. 12. For each duplex, the firststrand sequence is listed on top and the second strand sequence below.Modifications are depicted as numbers and are as follows: GN indicatesconjugation to a GalNAc linker in accordance with FIG. 8a . The sequenceand modification of STS12 (GN-TMPRSS6-hcm9) is also depicted in FIG. 7.

Example 9

Different modification variants of one GalNAc conjugated sequencetargeting TMPRSS6 (STS012) reduce TMPRSS6 expression in mouse primaryhepatocytes. For receptor-mediated uptake, cells were incubated with100, 10, 1 and 0.1 nM siRNA conjugate for 24 hours. Total RNA wasextracted and TMPRSS6 mRNA levels were quantified by Taqman qRT-PCR.TMPRSS6 mRNA levels were normalized to Actin mRNA levels. Mean+/−SD ofeach three technical replicates are shown in FIG. 10.

Example 10

Different modification variants of one GalNAc-conjugated sequencetargeting TMPRSS6 (STS012, GN-TMPRSS6-hcm9) were tested in vivo. 1 mg/kgand 3 mg/kg GalNAc-siRNA conjugate were subcutaneously injected intomale C57BL/6JOlaHsd mice. 14 days after treatment, TMPRSS6 (A) and HAMP(B) mRNA levels in the liver were analyzed by Taqman qRT-PCR. Barsrepresent mean of at least 4 animals+/−SD. Results are shown in FIG. 11.

Example 11

Two different modification variants of one GalNAc-conjugated sequencetargeting TMPRSS6 (STS012) were tested in vivo. 1 mg/kg GalNAc-siRNAconjugates were subcutaneously injected into male C57BL/6JOlaHsd mice.14 and 28 days after treatment, TMPRSS6 mRNA levels in the liver wereanalyzed by Taqman qRT-PCR (A). In addition, serum iron levels wereanalyzed (B). Results are shown in FIG. 13. Box plots represent medianof 4 animals. Statistical analysis is based on Kruskal-Wallis test withDunn's multiple comparison test against PBS group.

The sequences are as in Example 10.

Example 12

Two different modification variants of one GalNAc-conjugated sequencetargeting TMPRSS6 (STS12009L4, GN2-TMPRSS6-hcm9) were tested in vivo. 1mg/kg and 0.3 mg/kg GalNAc-siRNA conjugates were subcutaneously injectedinto male C57BL/6JOlaHsd mice. 14 days after treatment, TMPRSS6 mRNAlevels in the liver were analyzed by Taqman qRT-PCR (A). In addition,serum iron levels were analyzed (B). Results are shown in FIG. 14. Boxplots represent median of 4 animals. Statistical analysis is based onKruskal-Wallis test with Dunn's multiple comparison test against PBSgroup.

The duplexes used are shown below in Table 5. All sequences correspondto SEQ ID NO:17 and SEQ ID NO:18

TABLE 5 Modification variants of GalNAc-conjugatedsequences targeting TMPRSS6. sequence and chemistry duplex ID(top: first strand, bottom: second strand, both 5′-3′) STS12009L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V2L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGN2-mUmCmAmCfCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU STS12009V8L4mA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mAGN2-mUmCmAmCfCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU GN2-LucmU(ps)fU(ps)mAfGmUfAmAfAmCfCmUfUmUfUmGfAmG(ps)fA(ps)mCGN2-fGmUfCmUfCmAfAmAfAmGfGmUfUmUfAmCfU(ps)mA(ps)fA mA, mU, mC, mG -2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate GN2= GalNAc structure according to FIG. 8B

Example 13

Different modification variants of one siRNA targeting TMPRSS6 weretested in human Hep3B cells. An siRNA targeting Luciferase was used asnon-targeting control. All siRNAs were transfected with 1 μg/ml Atufectat 1 nM and 0.1 nM. Total RNA was extracted 48 h after transfection andTMPRSS6 mRNA levels were quantified by Taqman qRT-PCR. TMPRSS6 mRNAlevels were normalized to PTEN mRNA levels. Results are shown in FIG.15. Each bar represents mean+/−SD of three technical replicates.

The sequences are shown in Table 6 below. All sequences correspond toSEQ ID NO:17 (top) and SEQ ID NO:18 (bottom).

TABLE 6 Different modification variants of one siRNA targeting TMPRSS6.sequence and chemistry duplex (top: first strand, bottom: second strand,ID both 5′-3′) TMP01 mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAfUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU TMP66mAfAmCfCmAmGfAmAmGmAmAmGmCfAmGmGmUmGmAmUmCmAmCmCmUfGmCfUmUmCmUmUmCmUmGmGmUmU TMP69fAfAfCfCfAmGfAfAfGfAmAfGfCfAmGfGfUfGfAfUmCfAfCfCfUfGfCfUfUfCmUfUmCfUfGfGfUfU TMP79fAfAmCfCfAmGfAfAfGfAmAfGfCfAmGfGmUmGmAmUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU TMP80fAfAmCmCfAmGfAfAfGfAmAfGfCfAmGfGmUmGmAmUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU TMP81fAfAmCfCfAmGfAfAfGfAmAfGmCfAmGfGmUmGmAmUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU mA, mU, mC, mG - 2′-OMe RNA fA,fU, fC, fG - 2′-F DNA (ps) - phosphorothioate

Example 14

Modification variants of a GalNAc-conjugated siRNA targeting TMPRSS6were tested in human Hep3B cells. 150,000 cells were seeded per 6-well.After 24 h, siRNA conjugates were transfected with 1 μg/ml Atufect at10, 1, 0.1, 0.01, and 0.001 nM (A) or 5, 0.5, 0.05, 0.005 nM (B). AGalNAc-siRNA against Luciferase was used as non-targeting control. TotalRNA was extracted 72 h after transfection and TMPRSS6 mRNA levels werequantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized toPTEN mRNA levels (A) or Actin mRNA levels (B). Results are shown in FIG.16. Each bar represents mean+/−SD of three technical replicates.

Sequences are shown in Table 7, below

TABLE 7Modification variants of a GalNAc-conjugated siRNA targeting TMPRSS6sequence and chemistry duplex ID(top: first strand, bottom: second strand, both 5′-3′) STS12009L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V27L4mA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mAGN2-mUmCmAmCmCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU STS12009V41L4mA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU mA, mU, mC, mG -2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate GN2= GalNAc structure according to FIG. 8B

Example 15

A modification variant of a GalNAc-conjugated sequence targeting TMPRSS6(STS12009L4) was tested in mouse primary hepatocytes. Forreceptor-mediated uptake, cells were incubated with 100, 25, 5, 1, 0.25and 0.05 nM siRNA conjugate for 24 h. A GalNAc-siRNA targeting anunrelated sequence (GN-TTR) and a non-targeting GalNAc-siRNA (GN-Luc)were used as controls. Total RNA was extracted and TMPRSS6 mRNA levelswere quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalizedto PTEN mRNA levels. Results are shown in FIG. 17. Mean+/−SD of eachthree technical replicates are shown.

Sequences are shown in Table 7, above.

Example 16

Different DNA- and LNA-containing variants of one siRNA targetingTMPRSS6 were tested in human Hep3B cells. Therefore, 150,000 cells wereseeded per 6-well. After 24 h, siRNAs were transfected with 1 μg/mlAtufect at 0.1 nM siRNA. Total RNA was extracted 48 h after transfectionand TMPRSS6 mRNA levels were quantified by Taqman qRT-PCR. TMPRSS6 mRNAlevels were normalized to Actin mRNA levels. Results are shown in FIG.18. Each bar represents mean+/−SD of three technical replicates.

Sequences are shown in Table 8, below. All sequences correspond to SEQID NO:17 (top) and SEQ ID NO:18 (bottom).

TABLE 8 Different DNA- and LNA-containing variants ofone siRNA targeting TMPRSS6. sequence and chemistry duplex(top: first strand, bottom: second ID strand, both 5′-3′) TMP01mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAfUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU TMP95mAfAmCmCmAmGmAmAmGmAmAmGmCfAmGmGmUmGmAfUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU TMP99mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAmUmCmAmCmCmUfGmCfUmUmCmUmUmCmUmGmGmUmU TMP112mA[A]mCmCmAmGmAmAmGmAmAmGmCfAmGmGmUmGmAfUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU TMP114mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAmUmCmAmCmCmU{G}mCfUmUmCmUmUmCmUmGmGmUmU TMP115mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAmUmCmAmCmCmUfGmC{U}mUmCmUmUmCmUmGmGmUmU TMP116mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAmUmCmAmCmCmU[G]mCfUmUmCmUmUmCmUmGmGmUmU TMP117mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAmUmCmAmCmCmUfGmC[U]mUmCmUmUmCmUmGmGmUmU mA, mU, mC, mG - 2′-OMe RNA fA,fU, fC, fG - 2′-F DNA [A], [T], [C], [G] - DNA {A}, {U}, {C}, {G} - LNA(ps) - phosphorothioate

Example 17

The influence of inverted A and G RNA nucleotides at terminal 3′positions was analyzed using an siRNA against TMPRSS6. TMP70 containsphosphorothioates at all termini, whereas TMP82 and TMP83 contain ivA(TMP82) and ivG (TMP83) at the 3′-end of the antisense and at the 3′-endof the sense. Both inverted nucleotides are present in addition to theterminal nucleotide of the respective strands and are linked via aphosphorothioate bond. A non-related siRNA (PTEN) and a non-targetingsiRNA (Luci) were included as controls. All tested variants showcomparable activity under the tested conditions.

The experiment was conducted in Hep3B cells. Cells were seeded at adensity of 150,000 cells per 6-well, transfected with 1 nM and 0.1 nMsiRNA and 1 μg/ml Atufect after 24 h and lysed after 48 h. Total RNA wasextracted and TMPRSS6 and Actin mRNA levels were determined by TaqmanqRT-PCR. Results are shown in FIG. 19. Each bar represents mean±SD fromthree technical replicates.

The sequences are shown below in Table 9. Sequences correspond to SEQ IDNO:17 (top) or SEQ ID NO:18 (bottom).

TABLE 9 An siRNA against TMPRSS6 including invertedRNA nucleotides at different positions. duplex sequence and chemistry ID(top: first strand, bottom: second strand, both 5′-3′) TMP70mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU TMP82mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA(ps)ivAfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU(ps)ivA TMP83mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA(ps)ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU(ps)ivG mA, mU, mC, mG -2′-OMe RNA fA, fU, fC, fG - 2′-F DNA ivA, ivG - inverted RNA (3′-3′)(ps) - phosphorothioate

Example 18

Different siRNA duplexes containing inverted RNA nucleotides at both3′-ends were tested for serum stability. TMP84-TMP87 contain invertedRNA in addition to the last nucleotide in the sense strand and insteadof the last nucleotide in the antisense strand. TMP88-TMP91 containinverted RNA in addition to the last nucleotide in the antisense strandand instead of the last nucleotide in the sense strand. All inverted RNAnucleotides substitute for terminally used phosphorothioates. In thedesign of TMP84-TMP87, ivA and ivG confer higher stability to the testedsequence than ivU and ivC (part A). In the design of TMP88-TMP91, thereis no influence of base identity on duplex stability (part B).

Results are shown in FIG. 20. “UT” indicates untreated samples. “FBS”indicates siRNA duplexes which were incubated at 5 μM finalconcentration with 50% FBS for 3 d, phenol/chloroform-extracted andprecipitated with Ethanol. Samples were analyzed on 20% TBEpolyacrylamide gels in native gel electrophoresis.

Sequences are show in the Table 10, below, and correspond to SEQ IDNO:17 (top) and SEQ ID NO:18 (bottom).

TABLE 10 Different siRNA duplexes containing inverted RNAnucleotides at both 3′-ends. duplex sequence and chemistry ID(top: first strand, bottom: second strand, both 5′-3′) TMP70mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU TMP84mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivAfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG TMP85mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivUfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG TMP86mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivCfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG TMP87mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG TMP88mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivA TMP89mA(ps)fA(ps)mCfCmAfGmAfAmGfArnAfGmCfAmGfGmUfGmA ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivU TMP90mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivC TMP91mA(ps)fA(ps)mCfCmAfGmAfAnnGfAmAfGmCfAmGfGmUfGmA ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivG mA, mU, mC, mG - 2′-OMeRNA fA, fU, fC, fG - 2′-F DNA ivA, ivU, ivC, ivG - inverted RNA (3′-3′)(ps) - phosphorothioate

Example 19

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalyzed using an siRNA against TMPRSS6. TMP70 containsphosphorothioates at all termini, whereas TMP84-TMP87 contain ivG at the3′-end of the sense strand. The inverted RNA nucleotide is present inaddition to the last nucleotide and substitutes for twophosphorothioates. At the antisense 3′-end, ivA (TMP84), ivU (TMP85),ivC (TMP86) and ivG (TMP87) were tested. These inverted RNA nucleotideswere added instead of the terminal nucleotide and substitute forphosphorothioates. A non-related siRNA (PTEN) and a non-targeting siRNA(Luci) were included as controls. All tested variants show comparableactivity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 1 nM and 0.1 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR.Results are shown in FIG. 21. Each bar represents mean±SD from threetechnical replicates.

The sequences are as in Table 10, above.

Example 20

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalyzed using an siRNA against TMPRSS6. TMP70 containsphosphorothioates at all termini, whereas TMP88-TMP91 contain ivG at the3′-end of the antisense strand. The inverted RNA nucleotide is presentin addition to the last nucleotide and substitutes for twophosphorothioates. At the sense 3′-end, ivA (TMP88), ivU (TMP89), ivC(TMP90) and ivG (TMP91) were tested. These inverted RNA nucleotides wereadded instead of the terminal nucleotide and substitute forphosphorothioates. A non-related siRNA (PTEN) and a non-targeting siRNA(Luci) were included as controls. All tested variants show comparableactivity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 1 nM and 0.1 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR.Results are shown in FIG. 22. Each bar represents mean±SD from threetechnical replicates.

Sequences are as shown in Table 10, above.

Example 21

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalyzed using a GalNAc-siRNA conjugate targeting TMPRSS6 in liposomaltransfections. STS12009-L4 contains phosphorothioates at allnon-conjugated termini, whereas the tested variants contain an invertedRNA nucleotide at the 3′-end of both sense and antisense strand. Theinverted RNA is present in addition to the last nucleotide andsubstitutes for two terminal phosphorothioates (STS12009V10-L4 and-V11-L4) or is used in addition to the terminal phosphorothioates(STS12009V29-L4 and STS12009V30-L4). Inverted A (STS12009V10-L4 and-V29-L4) and inverted G (STS12009V11-L4 and -V30-L4) were used. Alltested variants show comparable activity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 5 nM to 0.0016 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR.Results are shown in FIG. 23. Each bar represents mean±SD of threetechnical replicates.

Sequences are set out in Table 11, below and correspond to SEQ ID NO:17(top) and SEQ ID NO:18 (bottom).

TABLE 11 An siRNA sequence including inverted RNA nucleotidesat the 3′ ends sequence and chemistry Duplex ID(top: first strand, bottom: second strand, both 5′-3′) STS12009L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V10L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAivAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfUivA STS12009V11L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAivGGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfUivG STS12009V29L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAivAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivA STS12009V30L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAivGGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivG mA, mU, mC, mG -2′-OMe RNA fA, fU, fC, fG - 2′-F DNA ivA, ivG - inverted RNA (3′-3′)(ps) - phosphorothioate GN2 = GalNAc structure according to FIG. 8B

Example 22

Serum stability assay of GalNAc-siRNA conjugates containing one PS2 atindividual ends. GalNAc was conjugated to the 5′-end of the sense strandand is internally stabilized by four PS. Phosphorodithioatemodifications were placed at the 5′-antisense (STS12009V37L4),3′-antisense (STS12009V36L4) and 3′-sense (STS12009V34L4) ends.STS12009L4 contains each two terminal PS at 5′-antisense, 3′-antisenseand 3′-sense ends, GalNAc is attached to the sense 5′-end and stabilizedby four internal PS. 5 μM GalNAc-siRNA conjugates were incubated with50% FBS for 3 d at 37° C. RNA was extracted and analyzed on 20% TBEpolyacrylamide gels. Results are shown in FIG. 24. “UT” indicatesuntreated samples, “FBS” indicates FBS treatment. “Control” indicates aless stabilized GalNAc-siRNA conjugate of different sequence.

Sequences are shown in Table 12, below, and correspond to SEQ ID NO:17(top) and SEQ ID NO:18 (bottom).

TABLE 12 GalNAc-siRNA conjugates containing one PS2(phosphorodithioate) at individual ends. sequence and chemistryduplex ID (top: first strand, bottom: second strand, both 5′-3′)STS12009L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V34L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU STS12009V36L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG(ps2)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V37L4mA(ps2)fAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU mA, mU, mC, mG -2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate (ps2) -phosphorodithioate GN2 = GalNAc structure according to FIG. 8B

Example 23

Activity of GalNAc-siRNA conjugates containing one PS2 at individualends. GalNAc was conjugated to the 5′-end of the sense strand and isinternally stabilized by four PS. Phosphorodithioate modifications wereplaced at the 5′-antisense (STS12009V37L4), 3′-antisense (STS12009V36L4)and 3′-sense (STS12009V34L4) ends. The experiment was conducted in mouseprimary hepatocytes. Cells were seeded at a density of 250,000 cells per6-well and treated with 100 nM, 10 nM and 1 nM GalNAc-siRNA.Transfections with 10 nM GalNAc-siRNA and 1 μg/ml Atufect served ascontrol. Cells were lysed after 24 h, total RNA was extracted andTMPRSS6 and PTEN mRNA levels were determined by Taqman qRT-PCR. Resultsare shown in FIG. 25. Each bar represents mean±SD from three technicalreplicates.

Sequences are as set out in Table 12, above.

Example 24

Serum stability assay of a GalNAc-siRNA conjugate (STS12009V40L4)containing each one PS2 at the second strand 5′-end and at the secondstrand 3′-end. GalNAc was conjugated to the 5′-end of the second strandand is not stabilized by any internal PS. STS12009L4 contains each twoterminal PS at 5′-antisense, 3′-antisense and 3′-sense ends, GalNAc isattached to the sense 5′-end and stabilized by four internal PS. 5 μMGalNAc-siRNA conjugates were incubated with 50% FBS for 3 d at 37° C.RNA was extracted and analyzed on 20% TBE polyacrylamide gels. Resultsare shown in FIG. 26. “UT” indicates untreated samples, “FBS” indicatesFBS treatment. “Control” indicates a less stabilized GalNAc-siRNAconjugate of different sequence.

Sequences are set out in Table 13, below, and are SEQ ID NO:17 (top) andSEQ ID NO:18 (bottom).

TABLE 13 GalNAc-siRNA conjugate containing each one PS2 at thesecond strand 5′-end and at the second strand 3′-end.sequence and chemistry duplex ID(top: first strand, bottom: second strand, both 5′-3′) STS12009L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V40L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAGNo-fU(ps2)mCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU mA, mU, mC, mG -2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate (ps2) -phosphorodithioate GN2 - GalNAc, structure according to FIG. 8B GNo -GN2 with phosphodiesters instead of (ps)

Example 25

Activity of a GalNAc-siRNA conjugate (STS12009V40L4) containing each onePS2 at the sense strand 5′-end and at the sense strand 3′-end. GalNAcwas conjugated to the 5′-end of the sense strand and is not stabilizedby any internal PS. STS12009L4 contains each two terminal PS at5′-antisense, 3′-antisense and 3′-sense ends, GalNAc is attached to thesense 5′-end and stabilized by four internal PS. The experiment wasconducted in mouse primary hepatocytes. Cells were seeded at a densityof 250,000 cells per 6-well and treated with 100 nM, 10 nM and 1 nMGalNAc-siRNA. A GalNAc conjugate of an siRNA against Luciferase(“GalNAc-Luc”) served as control. Cells were lysed after 24 h, total RNAwas extracted and TMPRSS6 and PTEN mRNA levels were determined by TaqmanqRT-PCR. Results are shown in FIG. 27. Each bar represents mean±SD fromthree technical replicates.

Sequences are as set out in Table 13, above.

Example 26

Inhibition of TMPRSS6 expression by different doses of GalNAc siRNAs inanimal model for hereditary hemochromatosis. HFE female mice (Herrmannet al., J. Mol. Med (Berl), 2004 82, 39-48) were treated subcutaneouslywith a single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate,respectively. Control groups were treated with PBS or with the nontargeting control GN2-Luc siRNA1 (GN2-Luc) by subcutaneous injection.Target gene expression in liver tissue was assessed by qRT PCR threeweeks after the injection of the conjugates.

Group mean and +/−SD. Statistics: Kruskal-Wallis test with uncorrectedDunn. Sequence and modification of siRNA conjugates are depicted in FIG.7. Results are shown in FIG. 28. P values: ****P<0.001; ***P<0.005;**0.01; *P<0.05.

Example 27

Increase of serum Hepcidin levels by different doses of GalNAc siRNAs inan animal model for hereditary hemochromatosis. HFE^(−/−) mice weretreated with single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate bysubcutaneous injection. Control groups were treated with PBS or with nontargeting control conjugate GN2-Luc siRNA 1 (GN2-Luc). Hepcidin levelswere determined in serum samples collected three weeks after injectionof the conjugates using ELISA kit (Intrinsic Life Science). Group meanswith SD. Kruskal-Wallis test with uncorrected Dunn's test againstcontrol group (GN2-Luc siRNA). Results are shown in FIG. 29. P values:****P<0.001; ***P<0.005; **0.01; *P<0.05.

Example 28

Reduction of serum iron levels by different doses of GalNAc siRNAs inanimal model for hereditary hemochromatosis. HFE^(−/−) mice were treatedwith single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate bysubcutaneous administration. Control groups were treated with PBS orwith non targeting control conjugate (GN2-Luc siRNA). Serum iron levelswere determined three weeks after the treatment. Group means+/−SD.Kruskal-Wallis test with uncorrected Dunn's test against control group(GN2-Luc). Sequences and modifications of siRNA conjugates are depictedin FIG. 7. Results are shown in FIG. 30. P values: ****<0.001;***P<0.005; **0.01; *P<0.05.

Example 29

Reduction of transferrin saturation by different doses of GalNAc siRNAsin animal model for hereditary hemochromatosis. HFE^(−/−) mice weretreated with single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate bysubcutaneous administration. Control groups were treated with PBS orwith non targeting control conjugate GN2-Luc siRNA1 (GN2-Luc). The %transferrin saturation in blood samples was determined three weeks afterthe treatment. Group means with SD. Kruskal-Wallis test with uncorrectedDunn's test against control group (GN2-Luc). Sequence and modificationof siRNA conjugates are depicted in FIG. 7. Results are shown in FIG.31. P values: ****P<0.001; ***P<0.005; **0.01; *P<0.05.

Example 30

Increase in Unsaturated Iron Binding Capacity (UIBC) in animal model forhereditary hemochromatosis. HFE^(−/−) mice were treated with single doseof 1 or 3 mg/kg of GalNAc siRNA conjugate by subcutaneousadministration. Control groups were treated with PBS or with nontargeting control conjugate GN2-Luc siRNA1 (GN2-LUC). Serum samples werecollected three weeks after treatment for determination of UIBC. Groupmeans with SD. Kruskal-Wallis test with uncorrected Dunn's test againstcontrol group (GN2-Luc). Results are shown in FIG. 32. P values:****P<0.001; ***P<0.005; **0.01; *P<0.05.

Example 31

Reduction of tissue iron levels by GalNAc siRNAs in animal model forhereditary hemochromatosis. HFE^(−/−) mice were treated with single doseof 1 or 3 mg/kg of GalNAc siRNA conjugate by subcutaneousadministration. Control groups were treated with PBS or with nontargeting control conjugate GN2-Luc siRNA1 (GN2-Luc). Iron levels inkidney tissue was assessed three weeks after the treatment. Box andWiskers (Tukey, median values) Kruskal-Wallis test with uncorrectedDunn's test against control group (GN2-Luc ). Results are shown in FIG.33.

Example 32

Reduction of TMPRSS6 mRNA expression by different siRNAs in Hep3B cells.8000 cells per well were plated in 96-well plates. The following daycells were transfected with 20 nM siRNA and 1 μg/ml AtuFECT. Two daysafter transfection cells were lysed and TMPRSS6 mRNA levels weredetermined by q-RT-PCR. TMPRSS6 mRNA levels were normalized toexpression levels of the house keeping gene ApoB. An siRNAs againstLuciferase was used as non targeting control. Average inhibition andstandard deviation of triplicate values relative to untreated cells areshown in FIG. 34. The sequences of the siRNAs are shown in Table 14,below.

TABLE 14 Sequences of siRNAs against TMPRSS6 tested in example 32.fU, fA, fC, fG - 2′F modified deoxynucleotides. mU, mA, mC, mG -2′O-methyl modified nucleotides. SEQ ID Duplex ID strand ID NO:siRNA sequence TMPRSS6- hcTMP-SR1-A 133mCfUmGfAmGfGmAfCmGfCmCfCmUfGmGfGmAfGmU SR1 hcTMP-SR1-B 134fAmCfUmCfCmCfAmGfGmGfCmGfUmCfCmUfCmAfG TMPRSS6- hcTMP-SR2-A 135mGfCmUfGmAfGmGfAmCfGmCfCmCfUmGfGmGfAmG SR2 hcTMP-SR2-B 136fCmUfCmCfCmAfGmGfGmCfGmUfCmCfUmCfAmGfC TMPRSS6- hcTMP-SR3-A 137mUfGmCfUmGfAmGfGmAfCmGfCmCfCmUfGmGfGmA SR3 hcTMP-SR3-B 138fUmCfCmCfAmGfGmGfCmGfUmCfCmUfCmAfGmCfA TMPRSS6- hcTMP-SR4-A 139mGfUmGfCmUfGmAfGmGfAmCfGmCfCmCfUmGfGmG SR4 hcTMP-SR4-B 140fCmCfCmAfGmGfGmCfGmUfCmCfUmCfAmGfCmAfC TMPRSS6- hcTMP-SR5-A 141mGfGmUfGmCfUmGfAmGfGmAfCmGfCmCfCmUfGmG SRS hcTMP-SR5-B 142fCmCfAmGfGmGfCmGfUmCfCmUfCmAfGmCfAmCfC TMPRSS6- hcTMP-SR6-A 143mGfGmGfUmGfCmUfGmAfGmGfAmCfGmCfCmCfUmG SR6 hcTMP-SR6-B 144fCmAfGmGfGmCfGmUfCmCfUmCfAmGfCmAfCmCfC TMPRSS6- hcTMP-SR8-A 145mCfGmGfGmGfUmGfCmUfGmAfGmGfAmCfGmCfCmC SR8 hcTMP-SR8-B 146fGmGfGmCfGmUfCmCfUmCfAmGfCmAfCmCfCmCfG TMPRSS6- hcTMP-SR9-A 147mAfCmGfGmGfGmUfGmCfUmGfAmGfGmAfCmGfCmC SR9 hcTMP-SR9-B 148fGmGfCmGfUmCfCmUfCmAfGmCfAmCfCmCfCmGfU TMPRSS6- hcTMP-SR10-A 149mUfAmCfGmGfGmGfUmGfCmUfGmAfGmGfAmCfGmC SR10 hcTMP-SR10-B 150fGmCfGmUfCmCfUmCfAmGfCmAfCmCfCmCfGmUfA TMPRSS6- hcTMP-SR11-A 151mGfUmAfCmGfGmGfGmUfGmCfUmGfAmGfGmAfCmG SR11 hcTMP-SR11-B 152fCmGfUmCfCmUfCmAfGmCfAmCfCmCfCmGfUmAfC TMPRSS6- hcTMP-SR12-A 153mAfGmUfAmCfGmGfGmGfUmGfCmUfGmAfGmGfAmC SR12 hcTMP-SR12-B 154fGmUfCmCfUmCfAmGfCmAfCmCfCmCfGmUfAmCfU TMPRSS6- hcTMP-SR13-A 155mAfAmGfUmAfCmGfGmGfGmUfGmCfUmGfAmGfGmA SR13 hcTMP-SR13-B 156fUmCfCmUfCmAfGmCfAmCfCmCfCmGfUmAfCmUfU TMPRSS6- hcTMP-SR15-A 157mGfGmAfAmGfUmAfCmGfGmGfGmUfGmCfUmGfAmG SR15 hcTMP-SR15-B 158fCmUfCmAfGmCfAmCfCmCfCmGfUmAfCmUfUmCfC TMPRSS6- hcTMP-SR16-A 158mGfGmGfAmAfGmUfAmCfGmGfGmGfUmGfCmUfGmA SR16 hcTMP-SR16-B 160fUmCfAmGfCmAfCmCfCmCfGmUfAmCfUmUfCmCfC TMPRSS6- hcTMP-SR17-A 161mGfGmGfGmAfAmGfUmAfCmGfGmGfGmUfGmCfUmG SR17 hcTMP-SR17-B 162fCmAfGmCfAmCfCmCfCmGfUmAfCmUfUmCfCmCfC TMPRSS6- hcTMP-SR18-A 163mUfGmGfGmGfAmAfGmUfAmCfGmGfGmGfUmGfCmU SR18 hcTMP-SR18-B 164fAmGfCmAfCmCfCmCfGmUfAmCfUmUfCmCfCmCfA TMPRSS6- hcTMP-SR19-A 165mCfUmGfGmGfGmAfAmGfUmAfCmGfGmGfGmUfGmC SR19 hcTMP-SR19-B 166fGmCfAmCfCmCfCmGfUmAfCmUfUmCfCmCfCmAfG TMPRSS6- hcTMP-SR21-A 167mAfGmCfUmGfGmGfGmAfAmGfUmAfCmGfGmGfGmU SR21 hcTMP-SR21-B 168fAmCfCmCfCmGfUmAfCmUfUmCfCmCfCmAfGmCfU TMPRSS6- hcTMP-SR22-A 169mUfAmGfCmUfGmGfGmGfAmAfGmUfAmCfGmGfGmG SR22 hcTMP-SR22-B 170fCmCfCmCfGmUfAmCfUmUfCmCfCmCfAmGfCmUfA TMPRSS6- hcTMP-SR23-A 171mGfUmAfGmCfUmGfGmGfGmAfAmGfUmAfCmGfGmG SR23 hcTMP-SR23-B 172fCmCfCmGfUmAfCmUfUmCfCmCfCmAfGmCfUmAfC TMPRSS6- hcTMP-SR24-A 173mAfGmUfAmGfCmUfGmGfGmGfAmAfGmUfAmCfGmG SR24 hcTMP-SR24-B 174fCmCfGmUfAmCfUmUfCmCfCmCfAmGfCmUfAmCfU TMPRSS6- hcTMP-SR26-A 175mGfUmAfGmUfAmGfCmUfGmGfGmGfAmAfGmUfAmC SR26 hcTMP-SR26-B 176fGmUfAmCfUmUfCmCfCmCfAmGfCmUfAmCfUmAfC TMPRSS6- hcTMP-SR27-A 177mAfGmUfAmGfUmAfGmCfUmGfGmGfGmAfAmGfUmA SR27 hcTMP-SR27-B 178fUmAfCmUfUmCfCmCfCmAfGmCfUmAfCmUfAmCfU TMPRSS6- hcTMP-SR28-A 179mGfAmGfUmAfGmUfAmGfCmUfGmGfGmGfAmAfGmU SR28 hcTMP-SR28-B 180fAmCfUmUfCmCfCmCfAmGfCmUfAmCfUmAfCmUfC TMPRSS6- hcTMP-SR29-A 181mCfGmAfGmUfAmGfUmAfGmCfUmGfGmGfGmAfAmG SR29 hcTMP-SR29-B 182fCmUfUmCfCmCfCmAfGmCfUmAfCmUfAmCfUmCfG TMPRSS6- hcTMP-SR30-A 183mGfCmGfAmGfUmAfGmUfAmGfCmUfGmGfGmGfAmA SR30 hcTMP-SR30-B 184fUmUfCmCfCmCfAmGfCmUfAmCfUmAfCmUfCmGfC TMPRSS6- hcTMP-SR31-A 185mGfGmCfGmAfGmUfAmGfUmAfGmCfUmGfGmGfGmA SR31 hcTMP-SR31-B 186fUmCfCmCfCmAfGmCfUmAfCmUfAmCfUmCfGmCfC TMPRSS6- hcTMP-SR32-A 187mGfGmGfCmGfAmGfUmAfGmUfAmGfCmUfGmGfGmG SR32 hcTMP-SR32-B 188fCmCfCmCfAmGfCmUfAmCfUmAfCmUfCmGfCmCfC TMPRSS6- hcTMP-SR33-A 189mGfGmGfGmCfGmAfGmUfAmGfUmAfGmCfUmGfGmG SR33 hcTMP-SR33-B 190fCmCfCmAfGmCfUmAfCmUfAmCfUmCfGmCfCmCfC TMPRSS6- hcTMP-SR34-A 191mUfGmGfGmGfCmGfAmGfUmAfGmUfAmGfCmUfGmG SR34 hcTMP-SR34-B 192fCmCfAmGfCmUfAmCfUmAfCmUfCmGfCmCfCmCfA TMPRSS6- hcTMP-SR35-A 193mUfUmGfGmGfGmCfGmAfGmUfAmGfUmAfGmCfUmG SR35 hcTMP-SR35-B 194fCmAfGmCfUmAfCmUfAmCfUmCfGmCfCmCfCmAfA TMPRSS6- TMPRSS6-hc-16A 195mUfAmUfUmCfCmAfAmAfGmGfGmCfAmGfCmUfGmA hc-16 TMPRSS6-hc-16B 196fUmCfAmGfCmUfGmCfCmCfUmUfUmGfGmAfAmUfA TMPRSS6- TMPRSS6-hc-17A 197mAfUmCfUmUfCmUfGmGfGmCfUmUfUmGfGmCfGmG hc-17 TMPRSS6-hc-17B 198fCmCfGmCfCmAfAmAfGmCfCmCfAmGfAmAfGmAfU TMPRSS6- TPRSS6-hc-18A 199mUfUmUfUmCfUmCfUmUfGmGfAmGfUmCfCmUfCmA hc-18 TMPRSS6-hc-18B 200fUmGfAmGfGmAfCmUfCmCfAmAfGmAfGmAfAmAfA TMPRSS6- TMPRSS6-hc-19A 201mGfAmAfUmAfGmAfCmGfGmAfGmCfUmGfGmAfGmU hc-19 TMPRSS6-hc-19B 202fAmCfUmCfCmAfGmCfUmCfCmGfUmCfUmAfUmUfC TMPRSS6- TMPRSS6-hc-21A 203mUfAmGfUmAfGmCfUmGfGmGfGmAfAmGfUmAfCmG hc-21 TMPRSS6-hc-21B 204fCmGfUmAfCmUfUmCfCmCfCmAfGmCfUmAfCmUfA TMPRSS6- TMPRSS6-hc-22A 205mAfGmAfUmCfCmUfGmGfGmAfGmAfAmGfUmGfGmC hc-22 TMPRSS6-hc-22B 206fGmCfCmAfCmUfUmCfUmCfCmCfAmGfGmAfUmCfU TMPRSS6- TMPRSS6-hc-23A 207mCfUmGfUmUfCmUfGmGfAmUfCmGfUmCfCmAfCmU hc-23 TMPRSS6-hc-23B 208fAmGfUmGfGmAfCmGfAmUfCmCfAmGfAmAfCmAfG TMPRSS6- TMPRSS6-hcmr-24A 209mCfUmCfAmCfCmUfUmGfAmAfGmGfAmCfAmCfCmU hcmr-24 TMPRSS6-hcmr-24B 210fAmGfGmUfGmUfCmCfUmUfCmAfAmGfGmUfGmAfG TMPRSS6- TMPRSS6-hcm-25 211mAfGmUfUmUfCmUfCmUfCmAfUmCfCmAfGmGfCmC hcm-25 TMPRSS6-hcm-25 212fGmGfCmCfUmGfGmAfUmGfAmGfAmGfAmAfAmCfU TMPRSS6- TMPRSS6-hcr-26A 213mGfUmAfCmCfCmUfAmGfGmAfAmAfUmAfCmCfAmG hcr-26 TMPRSS6-hcr-26B 214fCmUfGmGfUmAfUmUfUmCfCmUfAmGfGmGfUmAfC TMPRSS6- TMPRSS6-hc-27A 215mCfUmGfUmUfGmAfCmUfGmUfGmGfAmCfAmGfCmA hc-27 TMPRSS6-hc-27B 216fUmGfCmUfGmUfCmCfAmCfAmGfUmCfAmAfCmAfG

Example 33

Dose-Response of siRNAs Against TMPRSS6 in Hep3B Cells.

8000 cells per well were plated in 96-well plates. The following daycells were transfected with siRNA in indicated concentrations (10 nM-0.0nM) an 1 μg/ml At T Two days after transfection cells were lysed andTMPRSS6 mRNA levels were determined by q-RT-PCR. TMPSS6 mRNA levels werenormalized to expression levels of the house keeping gene Apo B. Averageinhibition and standard deviation of triplicate values relative to cellstreated with 100 nM of anon-targeting Luciferase-control siRNA are shownin FIG. 3. Table 15, blow, shows maximum inhibition, 105 and 95%confidence interval according to dose-response curves shown in FIG. 35.

TABLE 15 95% kd at Max confidence 20 nM as inhi- IC50 interval shown inDuplex ID bition [nM] [nM] FIG. 34 sd TMPRSS6-hcmr-24 73% 0.8 0.1-6.2 70% 2% TMPRSS-hc-17 82% 2.4 1.1-5.0  79% 5% TMPRSS-SR-27 94% 2.80.7-10.8 84% 4% TMPRSS-hcr-26 100%  3.5 1.4-9.0  98% 2% TMPRSS-hc-18 95%4.6 1.5-14.1 90% 1% TMPRSS-hc-23 83% 4.6 1.9-11.1 70% 7% TMPRSS-hcm-2587% 4.6 2.1-10.2 85% 3% TMPRSS-SR-21 100%  5.8 0.9-36.9 92% 4%TMPRSS-hc-19 100%  6.1 3.0-12.5 89% 2% TMPRSS-hc-16 100%  7.7 2.6-22.691% 3% TMPRSS-hc-22 99% 8.1 2.1-31.1 73% 20%  TMPRSS-SR-16 100%  8.42.5-28.2 88% 2% TMPRSS-SR-5 100%  8.4 2.4-29.0 85% 2%

Table 15: Maximum inhibition, IC50 and 95% confidence interval accordingto dose response shown in FIG. 35.

Example 34

Inhibition of TMPSS6 mRNA expression by receptor mediated uptake in 1°human hepatocytes.

Primary human hepatocytes were plated on collagen coated dishes andincubated with siRNA conjugates diluted in cell culture medium atconcentrations of 300 nM to 1 nM as indicated. 24 hours after exposingthe cells to siRNA conjugates total RNA was extracted and TMPRSS6expression was quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels werenormalized to Actin mRNA levels and to target mRNA levels of cellstreated with non targeting control siRNA conjugate GN2-Luc siRNA 1(GN2-Luc). Dose dependent inhibition of TMPRSS6 expression was observedby the GalNAc-TMPRSS6 siRNA conjugate.

Sequences and modifications of the conjugates are depicted in FIG. 7.Results are shown in FIG. 36.

Example 35

GalNAc TMPRSS6 siRNA Raises Hematocrit Values in Rodent Model forβ-Thalassemia Intermedia.

Hbb^(th3/+) mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) weretreated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6-hcm9(GN2-TMP), GN2-Luc siRNA 1 (GN2-Luc) or PBS as non targeting control oras vehicle control, respectively. On d 36 whole blood was collected intoheparin coated tubes for full blood examination. Hbb^(th3/+) mice wereobtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on aC57BL/6 background. Blood samples from untreated wild type (WT) mice(C57BL/6) were collected and analysed for comparisons. Scatter dot blot,mean+/−SD; n=3-6. Statistics: unpaired t test with Welch's correction.Results are shown in FIG. 37.

Example 36

GalNac TMPRSS6 siRNA Reduces Red Blood Cell Distribution Width in RodentModel for B-Thalassemia Intermedia.

Hbb^(th3/+) mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) weretreated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6-hcm9(GN2-TMP), GN2-Luc siRNA1 (GN2-Luc) or PBS as non targeting control oras vehicle control, respectively. On d 36 whole blood was collected intoheparin coated tubes for full blood examination. The Hbb^(th3/+) micewere obtained from Jackson Laboratory (Bar Harbor, Me.) and maintainedon a C57BL/6 background. Blood samples from untreated wild type (WT)mice (C57BL/6) were collected and analysed for comparisons. Scatter dotblot, mean+/−SD; n=3-6. Statistics: unpaired t test with Welch'scorrection. Results are shown in FIG. 38.

Examples 37

GalNAc TMPRSS6 siRNA reduces the proportion of reticulocytes in rodentmodel for β-thalassemia intermedia.

Hbb^(th3/+) mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) weretreated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6 hcm9(GN2-TMP), GN2-Luc siRNA 1 (GN2-Luc) or PBS as non targeting control oras vehicle control, respectively. On d 36 whole blood was collected intoheparin coated tubes for full blood examination. The Hbb^(th3/+) micewere obtained from Jackson Laboratory (Bar Harbor, Me.) and maintainedon a C57BL/6 background. Blood samples from untreated wild type (WT)mice (C57BL/6) were collected and analysed for comparisons. Scatter dotblot, mean+/−SD; n=3-6. Statistics: unpaired t test with Welch'scorrection. Results are shown in FIG. 39.

Examples 38

GalNAc TMPRSS6 siRNA reduces the amount of reactive oxygen species (ROS)in rodent model for β-thalassemia intermedia. Hbb^(th3/+) mice (Th3/+;Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and ond15 subcutaneously with 3 mg/kg GN2-TMPRSS6 hcm9 (GN2-TMP) or GN2-Luc 1siRNA (GN2-Luc) as non targeting control. On d 36 whole blood wascollected into heparin coated tubes for full blood examination. ROSmeasurements were performed 5 min. after addition of 2′7′dichloro-fluorescein as indicator (Siwaponanan et al, 2017 Blood, 129,3087-3099). Blood samples from GN2-TMPRSS6 hcm9 treated mice weremeasured twice. The Hbb^(th3/+) mice (Th3/+) were obtained from JacksonLaboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background.Blood samples from untreated wild type (WT) mice (C57BL/6) werecollected and analysed for comparisons. Scatter dot blot, mean+/−SD;n=4-6. Statistic: unpaired t test with Welch's correction. Results areshown in FIG. 40.

Example 39

GalNAc TMPRSS6 siRNA raises hemoglobin levels in rodent model forβ-thalassemia intermedia. Hbb^(th3/+) mice (Yang et al. 1995, PNAS Vol.92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3mg/kg GN2-TMPRSS6-hcm9 (GN2-TMP), GN2-Luc-siRNA 1 (GN2-Luc) or PBS asnon targeting control or as vehicle control, respectively. On d 36 wholeblood was collected into heparin coated tubes for full bloodexamination. Hbb^(th3/+) mice were obtained from Jackson Laboratory (BarHarbor, Me.) and maintained on a C57BL/6 background. Blood samples fromuntreated wild type (wt) mice (C57BL/6) were collected and analysed forcomparisons. Scatter dot blot, mean+/−SD; n=5-7. Statistics: Welch'st-tests uncorrected for multiple comparison. Results are shown in FIG.41. siRNA conjugates are depicted in FIG. 7.

Example 40

GalNAc TMPRSS6 reduces splenomegaly in rodent model for β-thalassemiaintermedia. Hbbt^(th3/+) mice (Yang et al. 1995, PNAS Vol. 92,11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kgGN2-TMPRSS6-hcm9 (GN2-TMP), GN2-Luc-siRNA 1 (GN2-Luc) or PBS as nontargeting control or as vehicle control, respectively. On d 39 spleenweights were assessed. Hbb^(th3/+) mice were obtained from JacksonLaboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background.Spleen weights from wild type (wt) mice (C57BL/6) treated with PBS wereassessed for comparisons. Scatter dot blot, mean+/−SD; n=5-7.Statistics: Welch's t-tests uncorrected for multiple comparison. Resultsare shown in FIG. 42. siRNA conjugates are depicted in FIG. 7.

Example 41

GalNAc TMPRSS6 improves red blood cell maturation in the bone marrow.Hbb^(th3/+) mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) weretreated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6-hcm9(GN2-TMP), GN2-Luc-siRNA 1 (GN2-Luc) or PBS as non targeting control oras vehicle control, on d39 cells were collected from the bone marrow andanalyzed by FACS analysis. Viable erythroid cells were separated intodistinct populations based on CD71, Ter119 and CD44 staining.Hbb^(th3/+) mice were obtained from Jackson Laboratory (Bar Harbor, Me.)and maintained on a C57BL/6 background. Erythroid cells from the bonemarrow of wild type (wt) mice (C57BL/6) treated with PBS were assessedfor comparisons. Bar graph, mean+/−SD; n=5-7. Statistics: Welch'st-tests uncorrected for multiple comparison. Results are shown in FIG.43. siRNA conjugates are depicted in FIG. 7.

Example 42

GalNAc TMPRSS6 reduces ineffective erythropoiesis in the spleen.Hbb^(th3/+) mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) weretreated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6 hcm9(GN2-TMP), GN2-Luc siRNA 1 (GN2-Luc) or PBS as non targeting control oras vehicle control, respectively. On d 39 cells were collected from thespleen and analyzed by FACS analysis. Viable erythroid cells wereseparated into distinct populations based on CD71, Ter119 and CD44staining. Hbb^(th3/+) mice were obtained from Jackson Laboratory (BarHarbor, Me.) and maintained on a C57BL/6 background. Erythroid cellsfrom the from wild type (wt) mice (C57BL/6) treated with PBS wereassessed for comparisons. Bar graph, mean+/−SD; n=5-7. Statistics:Welch's t-tests uncorrected for multiple comparison. Results are shownin FIG. 44. siRNA conjugates are depicted in FIG. 7.

Example 43

Reduction of TMPRSS6 Expression in Human Hepatocytes by GalNAc siRNAConjugates.

30,000 cpw human primary hepatocytes were seeded in collagen-coated96-well plates. Cell were treated with indicated amounts of siRNAconjugates immediately after plating. Cells were lysed 24 hours posttreatment and TMPRSS6 mRNA expression analyzed by TaqMan qRT-PCR.Triplicate values of TMPRSS6 normalized to ApoB (housekeeper) and tomean of untreated cells are shown.

Results are shown in FIG. 45a, 45b and FIG. 46, and sequences are shownin FIG. 47. The GN3 linker described is shown in FIG. 8C.

Example 44

Serum stability assay of GalNAc-siRNA conjugates with phosphorothioates,phosphorodithioates and phosphodiesters in terminal positions and in theGalNAc moiety. GalNAc was conjugated to the 5′-end of the sense strandand is internally stabilized by four PS (STS12009L4) or not, thenphosphodiester linkages are used instead (STS12009V54L50—V57L50).Phosphorodithioate modifications were placed at all terminal positionsof the duplex except of the first strand 5′-end (−V54L50), at the3′-ends only (−V55L50, −V57L50) or at the 3′-end of the second strandonly (−V56L50). In certain designs, phosphodiesters were used interminal positions of the siRNA duplex (−V56L50, −V57L50). 5 μMGalNAc-siRNA conjugates were incubated with 50% FBS for 3 d at 37° C.RNA was extracted and analyzed on 20% TBE polyacrylamide gels. “UT”indicates untreated samples, “FBS” indicates FBS treatment. “Control”indicates a less stabilized GalNAc-siRNA conjugate of differentsequence.

GalNAc conjugates of an siRNA targeting TMPRSS6 containing different endstabilization chemistries (phosphorothioate, phosphorodithioate,phosphodiester) were tested by receptor-mediated uptake in primary mousehepatocytes. GalNAc was conjugated to the 5′-end of the second strandand is internally stabilized by four PS (STS12009L4) or not, thenphosphodiester linkages are used instead (STS12009V54L50—V57L50).Phosphorodithioate modifications were placed at all terminal positionsof the duplex except of the first strand 5′-end (−V54L50), at the3′-ends only (−V55L50, −V57L50) or at the 3′-end of the second strandonly (−V56L50). In certain designs, phosphodiesters were used interminal positions of the siRNA duplex (−V56L50, −V57L50). Theexperiment was conducted in mouse primary hepatocytes. Cells were seededat a density of 20,000 cells per 96-well and treated with 125 nM to 0.2nM GalNAc-siRNA. Cells were lysed after 24 h, total RNA was extractedand TMPRSS6 and PTEN mRNA levels were determined by Taqman qRT-PCR. Eachbar represents mean±SD of three technical replicates.

Results and relevant sequences are shown in FIGS. 48-50.

Example 45

Reduction of TMPRSS6 Expression in Primary Murine Hepatocytes by GalNAcsiRNA Conjugates with 2′-O-Methyl-Uridine or5′-(E)-Vinylphosphonate-2′-O-Methyl-Uridine Replacing the2′-O-Methyl-Adenin at the 5′ Position of the First Strand.

Murine primary hepatocytes were seeded into collagen pre-coated 96 wellplates (Thermo Fisher Scientific, #A1142803) at a cell density of 30,000cells per well and treated with siRNA-conjugates at concentrationsranging from 100 nM to 0.1 nM. 24 h post treatment cells were lysed andRNA extracted with InviTrap® RNA Cell HTS 96 Kit/C24×96 preps (Stratec#7061300400) according to the manufactures protocol. Transcripts levelsof TMPRSS6 and housekeeping mRNA (Ptenll) were quantified by TaqMananalysis.

siRNA Conjugates:

first strand/ siRNA second duplex strand sequence & modificationSTS12009L4 TMPRSS6-mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG (X0027) hcm9-AfG mU (ps) fG (ps) mA TMPRSS6-GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm9-BL4fG (ps) mU (ps) fU STS12209V4L4 TMPRSS6-vinylphosphonate-mU (ps) fA (ps) mC fC mA fG mA fA mG fA (X0204)hcm209AV4 mA fG mC fA mG fG mU (ps) fG (ps) mA TMPRSS6-GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm209-BL4fG (ps) mU (ps) fA STS12209V5L4 TMPRSS6-vinylphosphonate-mU fA mC fC mA fG mA fA mG fA mA fG (x0205) hcm209-AV5mC fA mG fG mU (ps) fG (ps) mA TMPRSS6-GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm209-BL4fG (ps) mU (ps) fA STS12209L4 TMPRSS6-mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG (x0207) hcm209AfG mU (ps) fG (ps) mA TMPRSS6-GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm209-BL4fG (ps) mU (ps) fA STS12209V1L4 TMPRSS6-mU fA mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (x0208) hcm9-AV1(ps) fG (ps) mA TMPRSS6-GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm209-BL4fG (ps) mU (ps) fA STS18001 STS18001AmU(ps)fC(ps)mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA(ps) (X0028) fC(ps)mGSTS18001BL4 GN2 fCmGfUmAfCmGfCmGfGmAfAmUfAmCfUmUfC (ps) mG (ps) fALegend: mN 2′-O-methyl ribonucleotide (e.g. mU - 2′-O-methyl Uracil) fN2′-fluoro ribonucleotide (e.g. fC - 2′-fluoro Cytidin) (ps)phosphorothioate vinylphosphonate vinyl-(E)-phosphonate GN2 strcutureaccording to FIG. 8B

TaqMan Primer and Probes

PTEN-2 CACCGCCAAATTTAACTGCAGA PTEN-2 AAGGGTTTGATAAGTTCTAGCTGT PTEN-2FAM-TGCACAGTATCCTTTTGAAGACCATAAC CCA-TAMRA hTMSS6:379U17CCGCCAAAGCCCAGAAG hTMSS6:475L21 GGTCCCTCCCCAAAGGAATAG hTMSS6:416U28FLFAM-CAGCACCCGCCTGGGAACTTACTACAAC- BHQ1

Legend:

FAM—6-carboxyfluorescein (fluorescent dye)

TAMRA—tetramethylrhodamine (quencher)

BHQ1—black hole quencher 1 (quencher)

In Vitro Dose Response

Target gene expression in primary murine hepatocytes 24 h followingtreatment with TMPRSS6-siRNA carrying vinyl-(E)-phosphonate 2′OMe-Uracilat the 5′-position of the anti-sense strand and two phosphorothioatelinkages between the first three nucleotides (STS12209V4L4),vinyl-(E)-phosphonate 2′OMe-Uracil at the 5′-position of the anti-sensestrand and phosphodiester bonds between the first three nucleotides(STS12209V5L4), carrying 2′-O-methyl-Uracil and two phosphorothioatelinkages between the first three nucleotides at the 5′-position(STS12209L4) or carrying 2′-O-methyl-Uracil and two phosphodiesterlinkages between the first three nucleotides at the 5′-position(STS12209V1L4) or ) or (STS12009L4) as reference or a non-targetingGalNAc-siRNA (STS18001) at indicated concentrations or left untreated(UT).

Results are shown in FIG. 51.

Serum Stability

Serum stability of siRNA conjugates incubated for 4 hours (4 h) or 3days (3 d) or left untreated (0 h) in 50% FCS at 37° C. Following RNAwas extracted by phenol/chlorophorm/isoamyl alcohol extraction.Degradation was visualized by TBE-Polyacrylamid-gel-electrophoresis andstaining RNA with SybrGold.

Results are shown in FIG. 52: Serum stability of siRNA-conjugates vs.less stabilized positive control for nuclease degradation.

Example 46

Reduction of transferrin saturation by TMPRSS6 siRNA treatment in animalmodel for hereditary hemochromatosis type 1. Three months old C57BL6/Jcongenic female HFE knock-out mice (−/−) and three months old femaleC57BL6/J wild type mice (+/+) were used in this study (Herrmann et al.,Mol. Med, 2004. 82 (1): p1-3). HFE −/− mice (n=8) were treated on Day 1(1×) or on Day 1 and on Day 22 (2×) subcutaneously with either PBS (PBS)or 3 mg/kg TMPRSS6 siRNA conjugate (TMP) or 3 mg/kg non-targeting siRNAconjugate (Luc). In addition, cohort 7 to 10 received Deferipronesupplied in drinking water at a concentration of 0.5 mg/ml. Cohorts 1and 2 were terminated on Day 1 (Predose) all other cohorts on Day 43 (6weeks after start of the treatment) to collect serum and tissue samplesfor analyses. Serum iron levels (SFBC) and unsaturated iron bindingcapacity UIBC were assessed by calorimetric methods. Transferrinsaturation (Tf Sat) was calculated using the following formula:TfSat=(SFBC/TIBC)×100. Transferrin saturation is elevated in 3 monthsold HFE −/− mice at start of the treatment phase (cohort 2, Predose).Treatment with TMPRSS6 siRNA conjugates alone or in combination withDeferiprone reduced transferrin saturation in this rodent model forhereditary hemochromatosis and iron overload.

Results are shown in FIG. 53.

siRNA Conjugates:

siRNA conjugate Single strands Sequence and chemistry TMP TMP-A Strand5′mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC SEQ ID NO: 217fA mG fG mU (ps) fG (ps) mA 3′ TMP-B Strand5′GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU SEQ ID NO: 218mG fG (ps) mU (ps) fU 3′ Luc Luc-A5′mU(ps)fC(ps)mGfAmAfGmUfAmUfUmCfCmGfCmGfUm A (ps) fC(ps) mG 3′ Luc-B5′GN2 fC mG fU mA fC mG fC mG fG mA fA mU fA mC fUmU fC (ps) mG (ps) fA 3′ Legend: fN (N = A, C, G, U) denotes 2′Fluoro,2′DeoxyNucleosides mN (N = A, C, G, U) denotes 2′O Methyl Nucleosides(ps) indicates a phosphorothioate linkage GN2 GalNAc structure as inFIG. 8B TMP-A is identical to SEQ ID NO: 111 TMP-B is identical to SEQID NO: 112, except evidently that GN has been replaced by GN2 TMP-A basesequence is identical to SEQ ID NO: 333 TMP-B base sequence is identicalto SEQ ID NO: 334 TMP-A modification pattern is identical to SEQ ID NO:17 TMP-B modification pattern is identical to SEQ ID NO: 18 Luc-A isidentical to SEQ ID NO: 109 Luc-B is identical to SEQ ID NO: 110 Luc-Abase sequence is identical to SEQ ID NO: 347 Luc-B base sequence isidentical to SEQ ID NO: 348 Luc-A modification pattern is identical toSEQ ID NO: 31 Luc-B modification pattern is identical to SEQ ID NO: 32

Example 47

Treatment with TMPRSS6 siRNAs enhances the reduction of tissue ironlevels by iron chelator. 3 months old HFE −/− (rodent model forhereditary hemochromatosis and iron overload) or wild type mice wereused for this study and treated as described in Example 46. Liver tissuesamples were collected to assess liver iron concentrations before startof the treatment (predose) and at the end of the study at Day 43 (6weeks after start of treatments). Liver tissue samples were dried andiron concentrations from dry weights were determined by acid extractionmethod. At the start of the study liver iron levels were alreadyelevated in HFE −/− compared to wild type control mice (Predose).Treatment of HFE −/− mice with TMPRSS6 siRNA conjugates in combinationwith Deferiprone enhanced the reduction of tissue iron levels by theiron chelator. In other words, the combined siRNA/iron chelatortreatment led to a much greater reduction of iron content in the liverthan was achieved by the treatment with the siRNA or the chelator alone(compare columns 5 and 9 or 6 and 10). This is surprising because thetreatment with the siRNA alone (compare column 3 with columns 5 and 6)or with the iron chelator alone (column 7) has little to no effect onthe liver iron content. The liver iron content is further reduced byadministration of multiple doses of the siRNA (compare columns 9 and10).

siRNA conjugates are as described in Example 46 and results are shown inFIG. 54.

Summary SEQUENCE TABLE SEQ ID NO Name Sequence (5′-3′) 1 TMPRSS6-hc-1A6181715172727184715 2 TMPRSS6-hc-1B 2647364545462646361 3 TMPRSS6-h-2A6154645272747282718 4 TMPRSS6-h-2B 3645354745452717261 5 TMPRSS6-h-3A6281546184546173748 6 TMPRSS6-h-3B 3748461727361726351 7 TMPRSS6-hc-4A5171846174537271847 8 TMPRSS6-hc-4B 4736454827461736462 9 TMPRSS6-h-5A6153636462728284627 10 TMPRSS6-h-5B 4517353545171818261 11 TMPRSS6-h-6A8164536184718173535 12 TMPRSS6-h-6B 2828463647361827163 13 TMPRSS6-h-7A6451816452645173728 14 TMPRSS6-h-7B 3548462715271636271 15TMPRSS6-hcmr-8A 5181637352846261637 16 TMPRSS6-hcmr-8B4816151735284816362 17 TMPRSS6-hcm-9A 6273646282647284546 18TMPRSS6-hcm-9B 1727354715351718451 19 TMPRSS6-hc-10A 526362737283818462520 TMPRSS6-hc-10B 2517363835484518152 21 TMPRSS6-hc-11A8151717172537284738 22 TMPRSS6-hc-11B 3847354825464646263 23TMPRSS6-hcm-12A 8361715354847151847 24 TMPRSS6-hcm-12B4736264737282646183 25 TMPRSS6-hc-13A 5363635482648182618 26TMPRSS6-hc-13B 3615363715372818182 27 TMPRSS6-hcmr-14A7272825454538273738 28 TMPRSS6-hcmr-14B 3848453827272535454 29TMPRSS6-hcmr-15A 5452737164826252736 30 TMPRSS6-hcmr-15B1845251537164845272 31 TMPRSS6-Luc-siRNA-1A 5382645251738381638 32TMPRSS6-Luc-siRNA-1B 3816383856252715382 33 TMPRSS6-PTEN-A5a6g5u7u6g7u8u8g5g8 34 TMPRSS6-PTEN-B c7a7c6c6g7u6g6a7u5a 35hTMPRSS6 (upper) ccgccaaagcccagaag 36 hTMPRSS6 (lower)ggTcccTccccaaaggaaTag 37 hTMPRSS6 (probe) cagcacccgccTgggaacTTacTacaac38 mTMPRSS6 (upper) cggcaccTaccTTccacTcTT 39 mTMPRSS6 (lower)TcggTggTgggcaTccT 40 mTMPRSS6 (probe) ccgagaTgTTTccagcTccccTgTTcTa 41h-Aktin (upper) gcaTgggTcagaaggaTTccTaT 42 h-Aktin (lower)TgTagaaggTgTggTgccagaTT 43 h-Aktin (probe) TcgagcacggcaTcgTcaccaa 44mAktin (upper) gTTTgagaccTTcaacacccca 45 mAktin (lower)gaccagaggcaTacagggaca 46 mAktin (probe) ccaTgTacgTagccaTccaggcTgTg 47PTEN (upper) caccgccaaaTTTaacTgcaga 48 PTEN (lower)aagggTTTgaTaagTTcTagcTgT 49 PTEN (probe) TgcacagTaTccTTTTgaagaccaTaaccca50 mHAMP (upper) ccTgTcTccTgcTTcTccTccT 51 mHAMP (lower)aaTgTcTgcccTgcTTTcTTcc 52 mHAMP (probe) TgagcagcaccaccTaTcTccaTcaaca 53TMPRSS6-hcmr-8B 4816151735284816362 54 TMPRSS6-h-2B 364535474545271726155 TMPRSS6-hcm-12B 4736264737282646183 56 TMPRSS6-h-7B3548462715271636271 57 TMPRSS6-hcm-9B 1727354715351718451 58TMPRSS6-hc-1B 2647364545462646361 59 TMPRSS6-hc-10B 251736383548451815260 TMPRSS6-hc-4B 4736454827461736462 61 TMPRSS6-h-3B 374846172736172635162 TMPRSS6-h-5B 4517353545171818261 63 TMPRSS6-hc-11B3847354825464646263 64 TMPRSS6-hc-13B 3615363715372818182 65TMPRSS6-hcmr-14B 3848453827272535454 66 TMPRSS6-h-6B 282846364736182716367 TMPJH01A 6273646282647284546 68 TMPJH01B 1727354715351718451 69TMPJH02A 2237242282243284142 70 TMPJH02B 1723314715355754815 71 TMPJH03A2273282646283248182 72 TMPJH03B 5363718351715354815 73 TMPJH04A2273282646683248182 74 TMPJH05A 2273242242643244542 75 TMPJH05B5727354315355754455 76 TMPJH06A 2277646242643244542 77 TMPJH07A2277686242643244542 78 TMPJH08A 2273242246687244542 79 TMPJH09A2273242682687244542 80 TMPJH10A 2273242242643284586 81 TMPJH11A2273242242643288586 82 TMPJH12A 2273242246687284586 83 TMPJH13A6277646246687284586 84 TMPJH13B 5767354315755758855 85 TMPJH14A2273282282283284182 86 TMPJH14B 5327318315315318415 87 TMPJH15A2273282282643284182 88 TMPJH16A 2237242242247244582 89 TMPJH16B1723314355311358411 90 TMPJH17A 2273282242643284586 91 TMPJH18A6277682242643288142 92 TMPJH19A 6277682242643288586 93 TMPJH20A2233282242643284142 94 TMPJH21A 2277282242643284586 95 TMPJH22A2273282642643284586 96 TMPJH23A 2273282282643284586 97 TMPJH24A2273282246643284586 98 TMPJH25A 2273282242683284586 99 TMPJH26A2273282242647284586 100 TMPJH27B 5727754715355754855 101 TMPJH28B1723314311351714411 102 TMPJH29B 5767354315355754455 103 TMPJH30B5727754315355754455 104 TMPJH31B 5727358315355754455 105 TMPJH32B5727354315755754455 106 TMPJH33B 5727354315355758455 107 GN-TTR-hc-Aucuugguuacaugaaauccca 108 GN-TTR-hc-B ugggauuucauguaaccaaga 109GN2-Luc-siRNA 1-A 5(ps)3(ps)826452517383816(ps)3(ps)8 110GN2-Luc-siRNA 1-B GN2-38163838462527153(ps)8(ps)2 111 STS012-A6(ps)2(ps)736462826472845(ps)4(ps)6 112 STS012-BGN-17273547153517184(ps)5(ps)1 113 STS012-1-A6(ps)2(ps)736462826472845(ps)4(ps)6 114 STS012-1-BGN-57677547153517588(ps)5(ps)5 115 STS012-2-A6(ps)2(ps)736462826472845(ps)4(ps)6 116 STS012-2-BGN-57673543157557588(ps)5(ps)5 117 STS012-3-A6(ps)2(ps)736462826472845(ps)4(ps)6 118 STS012-3-BGN-57A7C547153517T8G(ps)5(ps)T 119 STS012-4-A6(ps)2(ps)776462466872845(ps)8(ps)6 120 STS012-4-BGN-57673543157557588(ps)5(ps)5 121 STS012-5-A6(ps)2(ps)736462426472845(ps)4(ps)6 122 STS012-5-BGN-17273543153517184(ps)5(ps)1 123 STS012-6-A6(ps)2(ps)73646242647284546(ps)7(ps)7 124 STS012-6-BGN-17273543153517184(ps)5(ps)1 125 STS012-7-A6(ps)2(ps)776866866472885(ps)8(ps)6 126 STS012-7-BGN-57677547153517588(ps)5(ps)5 127 STS012-8-A6(ps)2(ps)776866866472885(ps)8(ps)6 128 STS012-8-BGN-57673543157557588(ps)5(ps)5 129 STS012-9-A2(ps)2(ps)772422422472445(ps)4(ps)2 130 STS012-9-BGN-57277547157557544(ps)5(ps)5 131 control siRNA A5(ps)1(ps)645262735151828(ps)2(ps)7 132 control siRNA BGN-45353626284515271(ps)6(ps)2 133 hcTMP-SR1-AmCfUmGfAmGfGmAfCmGfCnnCfCmUfGmGfGmAfGmU 134 hcTMP-SR1-BfAmCfUmCfCmCfAmGfGmGfCmGfUmCfCmUfCmAfG 135 hcTMP-SR2-AmGfCmUfGmAfGmGfAmCfGmCfCmCfUmGfGmGfAmG 136 hcTMP-SR2-BfCmUfCmCfCmAfGmGfGmCfGmUfCmCfUmCfAmGfC 137 hcTMP-SR3-AmUfGmCfUmGfAmGfGmAfCmGfCmCfCmUfGmGfGmA 138 hcTMP-SR3-BfUmCfCmCfAmGfGmGfCmGfUmCfCmUfCmAfGmCfA 139 hcTMP-SR4-AmGfUmGfCmUfGmAfGmGfAmCfGmCfCmCfUmGfGmG 140 hcTMP-SR4-BfCmCfCmAfGmGfGmCfGmUfCmCfUmCfAmGfCmAfC 141 hcTMP-SR5-AmGfGmUfGmCfUmGfAmGfGmAfCmGfCmCfCmUfGmG 142 hcTMP-SR5-BfCmCfAmGfGmGfCmGfUmCfCmUfCmAfGmCfAmCfC 143 hcTMP-SR6-AmGfGmGfUmGfCmUfGmAfGmGfAmCfGmCfCmCfUmG 144 hcTMP-SR6-BfCmAfGmGfGmCfGmUfCmCfUmCfAmGfCmAfCmCfC 145 hcTMP-SR8-AmCfGmGfGmGfUmGfCmUfGmAfGmGfAmCfGmCfCmC 146 hcTMP-SR8-BfGmGfGmCfGmUfCmCfUmCfAmGfCmAfCmCfCmCfG 147 hcTMP-SR9-AmAfCmGfGmGfGmUfGmCfUmGfAmGfGmAfCmGfCmC 148 hcTMP-SR9-BfGmGfCmGfUmCfCmUfCmAfGmCfAmCfCmCfCmGfU 149 hcTMP-SR10-AmUfAmCfGmGfGmGfUmGfCmUfGmAfGmGfAmCfGmC 150 hcTMP-SR10-BfGmCfGmUfCmCfUmCfAmGfCmAfCmCfCmCfGmUfA 151 hcTMP-SR11-AmGfUmAfCmGfGmGfGmUfGmCfUmGfAmGfGmAfCmG 152 hcTMP-SR11-BfCmGfUmCfCmUfCmAfGmCfAmCfCmCfCmGfUmAfC 153 hcTMP-SR12-AmAfGmUfAmCfGmGfGmGfUmGfCmUfGmAfGmGfAmC 154 hcTMP-SR12-BfGmUfCmCfUmCfAmGfCmAfCmCfCmCfGmUfAmCfU 155 hcTMP-SR13-AmAfAmGfUmAfCmGfGmGfGmUfGmCfUmGfAmGfGmA 156 hcTMP-SR13-BfUmCfCmUfCmAfGmCfAmCfCmCfCmGfUmAfCmUfU 157 hcTMP-SR15-AmGfGmAfAmGfUmAfCmGfGmGfGmUfGmCfUmGfAmG 158 hcTMP-SR15-BfCmUfCmAfGmCfAmCfCmCfCmGfUmAfCmUfUmCfC 159 hcTMP-SR16-AmGfGmGfAmAfGmUfAmCfGmGfGmGfUmGfCmUfGmA 160 hcTMP-SR16-BfUmCfAmGfCmAfCmCfCmCfGmUfAmCfUmUfCmCfC 161 hcTMP-SRI7-AmGfCmGfGmAfAmGfUmAfCmGfGmGfGmUfGmCfUmG 162 hcTMP-SR17-BfCmAfGmCfAmCfCmCfCmGfUmAfCmUfUmCfCmCfC 163 hcTMP-SR18-AmUfGmGfGmGfAmAfGmUfAmCfGmGfGmGfUmGfCmU 164 hcTMP-SR18-BfAmGfCmAfCmCfCmCfGmUfAmCfUmUfCmCfCmCfA 165 hcTMP-SR19-AmCfUmGfGmGfGmAfAmGfUmAfCmGfGmGfGmUfGmC 166 hcTMP-SR19-BfGmCfAmCfCmCfCmGfUmAfCmUfUmCfCmCfCmAfG 167 hcTMP-SR21-AmAfGmCfUmGfGmGfGmAfAmGfUmAfCmGfGmGfGmU 168 hcTMP-SR21-BfAmCfCmCfCmGfUmAfCmUfUmCfCmCfCmAfGmCfU 169 hcTMP-SR22-AmUfAmGfCmUfGmGfGmGfAmAfGmUfAmCfGmGfGmG 170 hcTMP-SR22-BfCmCfCmCfGmUfAmCfUmUfCmCfCmCfAmGfCmUfA 171 hcTMP-SR23-AmGfUmAfGmCfUmGfCmGfGmAfAmGfUmAfCmGfGmG 172 hcTMP-SR23-BfCmCfCmGfUmAfCmUfUmCfCmCfCmAfGmCfUmAfC 173 hcTMP-SR24-AmAfGmUfAmGfCmUfGmGfGmGfAmAfGmUfAmCfGmG 174 hcTMP-SR24-BfCmCfGmUfAmCfUmUfCmCfCmCfAmGfCmUfAmCfU 175 hcTMP-SR26-AmGfUmAfGmUfAmGfCmUfGmGfGmGfAmAfGmUfAmC 176 hcTMP-SR26-BfGmUfAmCfUmUfCmCfCmCfAmGfCmUfAmCfUmAfC 177 hcTMP-SR27-AmAfGmUfAmGfUmAfGmCfUmGfGmGfGmAfAmGfUmA 178 hcTMP-SR27-BfUmAfCmUfUmCfCmCfCmAfGmCfUmAfCmUfAmCfU 179 hcTMP-SR28-AmGfAmGfUmAfGmUfAmGfCmUfGmGfGmGfAmAfGmU 180 hcTMP-SR28-BfAmCfUmUfCmCfCmCfAmGfCmUfAmCfUmAfCmUfC 181 hcTMP-SR29-AmCfGmAfGmUfAmGfUmAfGmCfUmGfGmGfGmAfAmG 182 hcTMP-SR29-BfCmUfUmCfCmCfCmAfGmCfUmAfCmUfAmCfUmCfG 183 hcTMP-SR30-AmGfCmGfAmGfUmAfGmUfAmGfCmUfGmGfGmGfAmA 184 hcTMP-SR30-BfUmUfCmCfCmCfAmGfCmUfAmCfUmAfCmUfCmGfC 185 hcTMP-SR31-AmGfGmCfGmAfGmUfAmGfUmAfGmCfUmGfGmGfGmA 186 hcTMP-SR31-BfUmCfCmCfCmAfGmCfUmAfCmUfAmCfUmCfGmCfC 187 hcTMP-SR32-AmGfGmGfCmGfAmGfUmAfGmUfAmGfCmUfGmGfGmG 188 hcTMP-SR32-BfCmCfCmCfAmGfCmUfAmCfUmAfCmUfCmGfCmCfC 189 hcTMP-SR33-AmGfGmGfGmCfGmAfGmUfAmGfUmAfGmCfUmGfGmG 190 hcTMP-SR33-BfCmCfCmAfGmCfUmAfCmUfAmCfUmCfGmCfCmCfC 191 hcTMP-SR34-AmUfGmGfGmGfCmGfAmGfUmAfGmUfAmGfCmUfGmG 192 hcTMP-SR34-BfCmCfAmGfCmUfAmCfUmAfCmUfCmGfCmCfCmCfA 193 hcTMP-SR35-AmUfUmGfGmGfGmCfGmAfGmUfAmGfUmAfGmCfUmG 194 hcTMP-SR35-BfCmAfGmCfUmAfCmUfAmCfUmCfGmCfCmCfCmAfA 195 TMPRSS6-hc-16AmUfAmUfUmCfCmAfAmAfGmGfGmCfAmGfCmUfGmA 196 TMPRSS6-hc-16BfUmCfAmGfCmUfGmCfCmCfUmUfUmGfGmAfAmUfA 197 TMPRSS6-hc-17AmA (ps) fU (ps) mCfUmUfCmUfGmGfGmCfUmUfUmGfGmC (ps) fG (ps) mG 198TMPRSS6-hc-17B GN3-fCmCfGmCfCmAfAmAfGmCfCmCfAmGfAmAfG (ps) mA (ps) fU199 TMPRSS6-hc-18AmU (ps) fU (ps) mUfUmCfUmCfUmUfGmGfAmGfUmCfCmU (ps) fC (ps) mA 200TMPRSS6-hc-18B GN3-fUmGfAmGfGmAfCmUfCmCfAmAfGmAfGmAfA (ps) mA (ps) fA201 TMPRSS6-hc-19A mGfAmAfUmAfGmAfCmGfGmAfGmCfUmGfGmAfGmU 202TMPRSS6-hc-19B fAmCfUmCfCmAfGmCfUmCfCmGfUmCfUmAfUmUfC 203 TMPRSS6-hc-21AmUfAmGfUmAfGmCfUmGfGmGfGmAfAmGfUmAfCmG 204 TMPRSS6-hc-21BfCmGfUmAfCmUfUmCfCmCfCmAfGmCfUmAfCmUfA 205 TMPRSS6-hc-22AmAfGmAfUmCfCmUfGmGfGmAfGmAfAmGfUmGfGmC 206 TMPRSS6-hc-22BfGmCfCmAfCmUfUmCfUmCfCmCfAmGfGmAfUmCfU 207 TMPRSS6-hc-23AmC (ps) fU (ps) mGfUmUfCmUfGmGfAmUfCmGfUmCfCmA (ps) fC (ps) mU 208TMPRSS6-hc-23B GN3-fAmGfUmGfGmAfCmGfAmUfCmCfAmGfAmAfC (ps) mA (ps) fG209 TMPRSS6-hcmr-24A mCfUmCfAmCfCmUfUmGfAmAfGmGfAmCfAmCfCmU 210TMPRSS6-hcmr-24B fAmGfGmUfGmUfCmCfUmUfCmAfAmGfGmUfGmAfG 211TMPRSS6-hcm-25AmA (ps) fG (ps) mUfUmUfCmUfCmUfCmAfUmCfCmAfGmG (ps) fC (ps) mC 212TMPRSS6-hcm-25B GN3-fGmGfCmCfUmGfGmAfUmGfAmGfAmGfAmAfA (ps) mC (ps) fU213 TMPRSS6-hcr-26AmG (ps) fU (ps) mAfCmCfCmUfAmGfGmAfAmAfUmAfCmC (ps) fA (ps) mG 214TMPRSS6-hcr-26B GN3-fCmUfGmGfUmAfUmUfUmCfCmUfAmGfGmGfU (ps) mA (ps) fC215 TMPRSS6-hc-27A mCfUmGfUmUfGmAfCmUfGmUfGmGfAmCfAmGfCmA 216TMPRSS6-hc-27B fUmGfCmUfGmUfCmCfAmCfAmGfUmCfAmAfCmAfG 217 STS12009L4-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA 218 STS12009L4-BGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU 219 STS12009V2L4-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA 220STS12009V2L4-B GN2-mUmCmAmCfCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU 221STS12009V8L4-A mA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mA222 STS12009V8L4-B GN2-mUmCmAmCfCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU223 GN2-Luc-A mU(ps)fU(ps)mAfGmUfAmAfAmCfCmUfUmUfUmGfAmG(ps)fA(ps)mC 224GN2-Luc-B GN2-fGmUfCmUfCmAfAmAfAmGfGmUfUmUfAmCfU(ps)mA(ps)fA 225 TMP01-AmAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA 226 TMP01-BfUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU 227 TMP66-AmAfAmCfCmAmGfAmAmGmAmAmGmCfAmGmGmUmGmA 228 TMP66-BmUmCmAmCmCmUfGmCfUmUmCmUmUmCmUmGmGmUmU 229 TMP69-AfAfAfCfCfAmGfAfAfGfAmAfGfCfAmGfGfUfGfA 230 TMP69-BfUmCfAfCfCfUfGfCfUfUfCmUfUmCfUfGfGfUfU 231 TMP79-AfAfAmCfCfAmGfAfAfGfAmAfGfCfAmGfGmUmGmA 232 TMP79-BmUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU 233 TMP80-AfAfAmCmCfAmGfAfAfGfAmAfGfCfAmGfGmUmGmA 234 TMP80-BmUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU 235 TMP81-AfAfAmCfCfAmGfAfAfGfAmAfGmCfAmGfGmUmGmA 236 TMP81-BmUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU 237 STS12009V27L4-AmA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mA 238STS12009V27L4-A GN2-mUmCmAmCmCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU 239STS12009V41L4-A mA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mA240 STS12009V41L4-A GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU241 TMP95-A mAfAmCmCmAmGmAmAmGmAmAmGmCfAmGmGmUmGmA 242 TMP95-BfUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU 243 TMP99-AmAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA 244 TMP99-BmUmCmAmCmCmUfGmCfUmUmCmUmUmCmUmGmGmUmU 245 TMP112-AmA[A]mCmCmAmGmAmAmGmAmAmGmCfAmGmGmUmGmA 246 TMP112-BfUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU 247 TMP114-AmAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA 248 TMP114-BmUmCmAmCmCmU{G}mCfUmUmCmUmUmCmUmGmGmUmU 249 TMP115-AmAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA 250 TMP115-BmUmCmAmCmCmUfGmC{U}mUmCmUmUmCmUmGmGmUmU 251 TMP116-AmAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA 252 TMP116-BmUmCmAmCmCmU[G]mCfUmUmCmUmUmCmUmGmGmUmU 253 TMP117-AmAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA 254 TMP117-BmUmCmAmCmCmUfGmC[U]mUmCmUmUmCmUmGmGmUmU 255 TMP70-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA 256 TMP70-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU 257 TMP82-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA(ps)ivA 258 TMP82-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU(ps)ivA 259 TMP83-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA(ps)ivG 260 TMP83-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU(ps)ivG 261 TMP84-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivA 262 TMP84-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG 263 TMP85-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivU 264 TMP85-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG 265 TMP86-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivC 266 TMP86-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG 267 TMP87-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivG 268 TMP87-BfU(ps)mC(ps)fAmCfCmUfGmCfUrnUfCmUfUmCfUmGfGmUfU ivG 269 TMP88-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG 270 TMP88-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivA 271 TMP89-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG 272 TMP89-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivU 273 TMP90-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG 274 TMP90-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivC 275 TMP91-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG 276 TMP91-BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivG 277 STS12009V10L4-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAivA 278 STS12009V10L4-BGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfUivA 279 STS12009V11L4-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAivG 280 STS12009V11L4-BGN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfUivG 281 STS12009V29L4-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAivA 282STS12009V29L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivA283 STS12009V30L4-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAivG 284STS12009V30L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivG285 STS12009V34L4-AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA 286STS12009V34L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU 287STS12009V36L4-A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG(ps2)mA 288STS12009V36L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU 289STS12009V37L4-A mA(ps2)fAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA 290STS12009V37L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU 291STS12009V40L4-A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA292 STS12009V40L4-B GNo-fU(ps2)mCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU293 STS12209V4L4-Avinylphosphonate-mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fGmC fA mG fG mU (ps) fG (ps) mA 294 STS12209V4L4-BGN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA295 STS12209V5L4-Avinylphosphonate-mU fA mC fC mA fG mA fA mG fA mA fG mC fA mG fGmU (ps) fG (ps) mA 296 STS12209V5L4-BGN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA297 STS12209L4-AmU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps)fG (ps) mA 298 STS12209L4-6GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA299 STS12209V1L4-AmU fA mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA 300STS12209V1L4-BGN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA301 STS18001-A mU(ps)fC(ps)mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA(ps)fC(ps)mG302 STS18001-B GN2 fCmGfUmAfCmGfCmGfGmAfAmUfAmCfUrnUfC (ps) mG (ps) fA303 PTEN-2-A caccgccaaaTTTaacTgcaga 304 PTEN-2-BaagggTTTgaTaagTTcTagcTgT 305 PTEN-2-CFAM-TgcacagTaTccTTTTgaagaccaTaaccca-TAMRA 306 hTMSS6: 379U17ccgccaaagcccagaag 307 hTMSS6: 475L21 ggTcccTccccaaaggaaTag 308hTMSS6: 416U28FL FAM-cagcacccgccTgggaacTTacTacaac-BHQ1 309STS12009V54L50-AmA (ps) fA (ps) mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG (ps2) mA 310STS12009V54L50-BGNo - fU (ps2) mCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU (ps2) fU 311STS12009V55L50-AmA (ps) fA (ps) mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG (ps2) mA 312STS12009V55L50-B GNo - fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU (ps2) fU 313STS12009V56L50-A mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA  314STS12009V56L50-B GNo - fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU (ps2) fU 315STS12009V57L50-A mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG (ps2) mA 316STS12009V57L50-B GNo - fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU (ps2) fU 317TMPRSS6-hc-1A augucuuucacacuggcuu 318 TMPRSS6-hc-1B aagccagugugaaagacau319 TMPRSS6-h-2A auugaguacacgcagacug 320 TMPRSS6-h-2Bcagucugcguguacucaau 321 TMPRSS6-h-3A aaguugauggugaucccgg 322TMPRSS6-h-3B ccgggaucaccaucaacuu 323 TMPRSS6-hc-4A uucuggaucguccacuggc324 TMPRSS6-hc-4B gccaguggacgauccagaa 325 TMPRSS6-h-5Aauucacagaacagaggaac 326 TMPRSS6-h-5B guuccucuguucugugaau 327TMPRSS6-h-6A guagucauggcuguccucu 328 TMPRSS6-h-6B agaggacagccaugacuac329 TMPRSS6-h-7A aguuguaguaaguucccag 330 TMPRSS6-h-7Bcugggaacuuacuacaacu 331 TMPRSS6-hcmr-8A uuguacccuaggaaauacc 332TMPRSS6-hcmr-8B gguauuuccuaggguacaa 333 TMPRSS6-hcm-9Aaaccagaagaagcagguga 334 TMPRSS6-hcm-9B ucaccugcuucuucugguu 335TMPRSS6-hc-10A uaacaacccagcguggaau 336 TMPRSS6-hc-10Bauuccacgcuggguuguua 337 TMPRSS6-hc-11A guuucucucauccaggccg 338TMPRSS6-hc-11B cggccuggaugagagaaac 339 TMPRSS6-hcm-12Agcaucuucugggcuuuggc 340 TMPRSS6-hcm-12B gccaaagcccagaagaugc 341TMPRSS6-hc-13A ucacacuggaaggugaaug 342 TMPRSS6-hc-13Bcauucaccuuccaguguga 343 TMPRSS6-hcmr-14A cacagaugugucgaccccg 344TMPRSS6-hcmr-14B cggggucgacacaucugug 345 TMPRSS6-hcmr-15Auguacccuaggaaauacca 346 TMPRSS6-hcmr-15B ugguauuuccuaggguaca 347TMPRSS6-Luc-siRNA-1A ucgaaguauuccgcguacg 348 TMPRSS6-Luc-siRNA-1Bcguacgcggaauacuucga 349 TMPRSS6-PTEN-A uaaguucuagcuguggugg 350TMPRSS6-PTEN-B ccaccacagcuagaacuua 351 GN-TTR-hc-Aucuugguuacaugaaauccc a 352 GN-TTR-hc-B ugggauuucauguaaccaag a 353STS012-6-A aaccagaagaagcaggugacc 354 control siRNA A uuaguaaaccuuuugagac355 control siRNA B gucucaaaagguuuacuaa 356 hcTMP-SR1-Acugaggacgcccugggagu 357 hcTMP-SR1-B acucccagggcguccucag 358 hcTMP-SR2-Agcugaggacgcccugggag 359 hcTMP-SR2-B cucccagggcguccucagc 360 hcTMP-SR3-Augcugaggacgcccuggga 361 hcTMP-SR3-B ucccagggcguccucagca 362 hcTMP-SR4-Agugcugaggacgcccuggg 363 hcTMP-SR4-B cccagggcguccucagcac 364 hcTMP-SR5-Aggugcugaggacgcccugg 365 hcTMP-SR5-B ccagggcguccucagcacc 366 hcTMP-SR6-Agggugcugaggacgcccug 367 hcTMP-SR6-B cagggcguccucagcaccc 368 hcTMP-SR8-Acggggugcugaggacgccc 369 hcTMP-SR8-B gggcguccucagcaccccg 370 hcTMP-SR9-Aacggggugcugaggacgcc 371 hcTMP-SR9-B ggcguccucagcaccccgu 372 hcTMP-SR10-Auacggggugcugaggacgc 373 hcTMP-SR10-B gcguccucagcaccccgua 374hcTMP-SR11-A guacggggugcugaggacg 375 hcTMP-SR11-B cguccucagcaccccguac376 hcTMP-SR12-A aguacggggugcugaggac 377 hcTMP-SR12-Bguccucagcaccccguacu 378 hcTMP-SR13-A aaguacggggugcugagga 379hcTMP-SR13-B uccucagcaccccguacuu 380 hcTMP-SR15-A ggaaguacggggugcugag381 hcTMP-SR15-B cucagcaccccguacuucc 382 hcTMP-SR16-Agggaaguacggggugcuga 383 hcTMP-SR16-B ucagcaccccguacuuccc 384hcTMP-SR17-A ggggaaguacggggugcug 385 hcTMP-SR17-B cagcaccccguacuucccc386 hcTMP-SR18-A uggggaaguacggggugcu 387 hcTMP-SR18-Bagcaccccguacuucccca 388 hcTMP-SR19-A cuggggaaguacggggugc 389hcTMP-SR19-B gcaccccguacuuccccag 390 hcTMP-SR21-A agcuggggaaguacggggu391 hcTMP-SR21-B accccguacuuccccagcu 392 hcTMP-SR22-Auagcuggggaaguacgggg 393 hcTMP-SR22-B ccccguacuuccccagcua 394hcTMP-SR23-A guagcuggggaaguacggg 395 hcTMP-SR23-B cccguacuuccccagcuac396 hcTMP-SR24-A aguagcuggggaaguacgg 397 hcTMP-SR24-Bccguacuuccccagcuacu 398 hcTMP-SR26-A guaguagcuggggaaguac 399hcTMP-SR26-B guacuuccccagcuacuac 400 hcTMP-SR27-A aguaguagcuggggaagua401 hcTMP-SR27-B uacuuccccagcuacuacu 402 hcTMP-SR28-Agaguaguagcuggggaagu 403 hcTMP-SR28-B acuuccccagcuacuacuc 404hcTMP-SR29-A cgaguaguagcuggggaag 405 hcTMP-SR29-B cuuccccagcuacuacucg406 hcTMP-SR30-A gcgaguaguagcuggggaa 407 hcTMP-SR30-Buuccccagcuacuacucgc 408 hcTMP-SR31-A ggcgaguaguagcugggga 409hcTMP-SR31-B uccccagcuacuacucgcc 410 hcTMP-SR32-A gggcgaguaguagcugggg411 hcTMP-SR32-B ccccagcuacuacucgccc 412 hcTMP-SR33-Aggggcgaguaguagcuggg 413 hcTMP-SR33-B cccagcuacuacucgcccc 414hcTMP-SR34-A uggggcgaguaguagcugg 415 hcTMP-SR34-B ccagcuacuacucgcccca416 hcTMP-SR35-A uuggggcgaguaguagcug 417 hcTMP-SR35-Bcagcuacuacucgccccaa 418 TMPRSS6-hc-16A uauuccaaagggcagcuga 419TMPRSS6-hc-16B ucagcugcccuuuggaaua 420 TMPRSS6-hc-17Aaucuucugggcuuuggcgg 421 TMPRSS6-hc-17B ccgccaaagcccagaagau 422TMPRSS6-hc-18A uuuucucuuggaguccuca 423 TMPRSS6-hc-18Bugaggacuccaagagaaaa 424 TMPRSS6-hc-19A gaauagacggagcuggagu 425TMPRSS6-hc-19B acuccagcuccgucuauuc 426 TMPRSS6-hc-21Auaguagcuggggaaguacg 427 TMPRSS6-hc-21B cguacuuccccagcuacua 428TMPRSS6-hc-22A agauccugggagaaguggc 429 TMPRSS6-hc-22Bgccacuucucccaggaucu 430 TMPRSS6-hc-23A cuguucuggaucguccacu 431TMPRSS6-hc-23B aguggacgauccagaacag 432 TMPRSS6-hcmr-24Acucaccuugaaggacaccu 433 TMPRSS6-hcmr-24B agguguccuucaaggugag 434TMPRSS6-hcm-25A aguuucucucauccaggcc 435 TMPRSS6-hcm-25Bggccuggaugagagaaacu 436 TMPRSS6-hcr-26A guacccuaggaaauaccag 437TMPRSS6-hcr-26B cugguauuuccuaggguac 438 TMPRSS6-hc-27Acuguugacuguggacagca 439 TMPRSS6-hc-27B ugcuguccacagucaacag 440STS12209V4L4-A uaccagaagaagcagguga 441 STS12209V4L4-Bucaccugcuucuucuggua 442 STS18001-A ucgaaguauuccgcguacg 443 STS18001-Bcguacgcggaauacuucga 444 PTEN-2-A caccgccaaaTTTaacTgcaga 445 PTEN-2-BaagggTTTgaTaagTTcTagcTgT 446 PTEN-2-C TgcacagTaTccTTTTgaagaccaTaaccca447 hTMSS6: 379U17 ccgccaaagcccagaag 448 hTMSS6: 475L21ggTcccTccccaaaggaaTag 449 hTMSS6: 416U28FL cagcacccgccTgggaacTTacTacaacKey 1 = 2′F-dU 2 = 2′dA 3 = 2′F-dC 4 = 2′F-dG 5 = 2′OMe-rU 6 = 2′OMe-rA7 = 2′OMe-rC 8 = 2′OMe-rG T = dT mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC,fG - 2′-F DNA (ps) - phosphorothioate GN = GalNAc linker GN according tofig. 8A GN2 = GalNAc structure according to fig. 8B GN3 = GalNAc linkerstructure according to fig. 80 GNo = GN2 with phosphodiesters instead of(ps) [A], [T], [C], [G] - DNA {A}, {U}, {C}, {G} - LNA ivA, ivC, ivU,ivG - inverted RNA (3′-3′) (ps2) - phosphorodithioate vinylphosphonatevinyl-(E)-phosphonate FAM - 6-Carboxyfluorescein TAMRA -5-Carboxytetramethylrhodamine BHQ - Black Hole Quencher 1

Where specific linkers and or modified linkages are taught within an RNAsequence, such as PS, PS2, GN, GN2, GN3 etc, these are optional parts ofthe sequence, but are a preferred embodiment of that sequence.

The following abbreviations may be used:

GN

GN2

GN3

GNo Same as GN2 but with phosphodiesters instead of phosphorothioatesST23 (building block for synthesis)

ST41/C4XLT (building block for synthesis)

ST43/C6XLT (building block for synthesis)

Long trebler/ltrb/STKS (building block for synthesis)

[ST23 (ps)]3 ST41 GN2 (see above) (ps) [ST23 (ps)]3 ST43 GN3 (see above)(ps) ST23(ps) long GN (see above) trebler(ps)

1. A combination comprising a nucleic acid for inhibiting expression ofTMPRSS6 and one or more iron chelators, wherein the nucleic acidcomprises at least one duplex region that comprises at least a portionof a first strand and at least a portion of a second strand that is atleast partially complementary to the first strand, wherein said firststrand is at least partially complementary to at least a portion of RNAtranscribed from the TMPRSS6 gene, wherein said first strand comprises anucleotide sequence selected from the following sequences: SEQ ID NOs:333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339, 341, 343,345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406,407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,436, 438, 440, or
 442. 2. A combination for use in the treatment ofhemochromatosis, porphyria cutanea tarda, blood disorders, such asβ-thalassemia or sickle cell disease, congenital dyserythropoieticanemia, marrow failure syndromes, myelodysplasia, transfusional ironoverload, a disorder associated with iron overload, Parkinson's Disease,Alzheimer's Disease or Friedreich's Ataxia associated with ironoverload, and infections and non-relapse related mortality associatedwith bone marrow transplantation, preferably of hemochromatosis, thecombination comprising a nucleic acid for inhibiting expression ofTMPRSS6 and one or more iron chelators, wherein the nucleic acidcomprises at least one duplex region that comprises at least a portionof a first strand and at least a portion of a second strand that is atleast partially complementary to the first strand, wherein said firststrand is at least partially complementary to at least a portion of RNAtranscribed from the TMPRSS6 gene, wherein said first strand comprises anucleotide sequence selected from the following sequences: SEQ ID NOs:333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339, 341, 343,345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406,407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,436, 438, 440, or
 442. 3. A nucleic acid for use in the treatment ofhemochromatosis, porphyria cutanea tarda, blood disorders, such asβ-thalassemia or sickle cell disease, congenital dyserythropoieticanemia, marrow failure syndromes, myelodysplasia, transfusional ironoverload, a disorder associated with iron overload, Parkinson's Disease,Alzheimer's Disease or Friedreich's Ataxia associated with ironoverload, and infections and non-relapse related mortality associatedwith bone marrow transplantation, preferably of hemochromatosis, whereinthe nucleic acid inhibits expression of TMPRSS6, wherein the nucleicacid comprises at least one duplex region that comprises at least aportion of a first strand and at least a portion of a second strand thatis at least partially complementary to the first strand, wherein saidfirst strand is at least partially complementary to at least a portionof RNA transcribed from the TMPRSS6 gene, wherein said first strandcomprises a nucleotide sequence selected from the following sequences:SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339,341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374,376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402,404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430,432, 434, 436, 438, 440, or 442, wherein the nucleic acid isadministered with one or more iron chelators.
 4. A kit comprising anucleic acid for inhibiting expression of TMPRSS6 and one or more ironchelators, wherein the nucleic acid comprises at least one duplex regionthat comprises at least a portion of a first strand and at least aportion of a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of RNA transcribed from the TMPRSS6gene, wherein said first strand comprises a nucleotide sequence selectedfrom the following sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325,327, 329, 331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362,364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,392, 394, 396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418,420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, or
 442. 5. Apharmaceutical composition comprising a nucleic acid for inhibitingexpression of TMPRSS6 and one or more iron chelators, wherein thenucleic acid comprises at least one duplex region that comprises atleast a portion of a first strand and at least a portion of a secondstrand that is at least partially complementary to the first strand,wherein said first strand is at least partially complementary to atleast a portion of RNA transcribed from the TMPRSS6 gene, wherein saidfirst strand comprises a nucleotide sequence selected from the followingsequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335,337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398,400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426,428, 430, 432, 434, 436, 438, 440, or 442, wherein the nucleic acid,iron chelator or both are combined with a physiologically acceptableexcipient.
 6. A pharmaceutical composition for use in the treatment ofhemochromatosis, porphyria cutanea tarda, blood disorders, such asβ-thalassemia or sickle cell disease, congenital dyserythropoieticanemia, marrow failure syndromes, myelodysplasia, transfusional ironoverload, a disorder associated with iron overload, Parkinson's Disease,Alzheimer's Disease or Friedreich's Ataxia associated with ironoverload, and infections and non-relapse related mortality associatedwith bone marrow transplantation, preferably of hemochromatosis, whereinthe pharmaceutical composition comprises a nucleic acid for inhibitingexpression of TMPRSS6 and one or more iron chelators, wherein thenucleic acid comprises at least one duplex region that comprises atleast a portion of a first strand and at least a portion of a secondstrand that is at least partially complementary to the first strand,wherein said first strand is at least partially complementary to atleast a portion of RNA transcribed from the TMPRSS6 gene, wherein saidfirst strand comprises a nucleotide sequence selected from the followingsequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335,337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398,400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426,428, 430, 432, 434, 436, 438, 440, or 442, wherein the nucleic acid,iron chelator or both are combined with a physiologically acceptableexcipient.
 7. The combination of claim 1, combination for use of claim2, nucleic acid for use of claim 3, kit of claim 4, composition of claim5 or composition for use of claim 6, wherein the second strand comprisesa nucleotide sequence of SEQ ID NO: 334, 318, 320, 322, 324, 326, 328,330, 332, 336, 338, 340, 342, 344, 346, 357,
 359. 361, 363, 365, 367,369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395,397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423,425, 427, 429, 431, 433, 435, 437, 439, 441, or
 443. 8. The combination,combination for use, nucleic acid for use, kit, composition orcomposition for use according to any preceding claim, wherein the one ormore iron chelators are selected from deferoxamine, deferiprone,deferasirox (Exjade) and deferairox (Jadenu), optionally wherein theiron chelator is deferiprone.
 9. The combination, combination for use,nucleic acid for use, kit, composition or composition for use accordingto any preceding claim, wherein one or more nucleotides on the firstand/or second strand are modified, to form modified nucleotides.
 10. Thecombination, combination for use, or nucleic acid for use according toclaim 9, wherein said first strand comprises a nucleotide sequence ofSEQ ID NO:17, and wherein said second strand comprises the nucleotidesequence of SEQ ID NO:18 SEQ ID 5′aaccagaagaagca 6273646282647284546NO: 17 gguga 3′ SEQ ID 5′ucaccugcuucuuc 1727354715351718451 NO: 18ugguu 3′

wherein the specific modifications are depicted by the following numbers1=2′F-dU, 2=2′F-dA, 3=2′F-dC, 4=2′F-dG, 5=2′-OMe-rU; 6=2′-OMe-rA;7=2′-OMe-rC; 8=2′-OMe-rG.
 11. A combination comprising a nucleic acidfor inhibiting expression of TMPRSS6 and deferiprone, wherein thenucleic acid comprises at least one duplex region that comprises atleast a portion of a first strand and at least a portion of a secondstrand that is at least partially complementary to the first strand,wherein said first strand is at least partially complementary to atleast a portion of RNA transcribed from the TMPRSS6 gene, wherein thefirst strand comprises the nucleotide sequence of SEQ ID NO:17 and thesecond strand comprises the nucleotide sequence of SEQ ID NO:18.
 12. Acombination for use in the treatment of hemochromatosis, porphyriacutanea tarda, blood disorders, such as β-thalassemia or sickle celldisease, congenital dyserythropoietic anemia, marrow failure syndromes,myelodysplasia, transfusional iron overload, a disorder associated withiron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich'sAtaxia associated with iron overload, and infections and non-relapserelated mortality associated with bone marrow transplantation,preferably hemochromatosis, comprising a nucleic acid for inhibitingexpression of TMPRSS6 and deferiprone, wherein the nucleic acidcomprises at least one duplex region that comprises at least a portionof a first strand and at least a portion of a second strand that is atleast partially complementary to the first strand, wherein said firststrand is at least partially complementary to at least a portion of RNAtranscribed from the TMPRSS6 gene, wherein the first strand comprisesthe nucleotide sequence of SEQ ID NO:17 and the second strand comprisesthe nucleotide sequence of SEQ ID NO:18.
 13. A nucleic acid for use inthe treatment of hemochromatosis, porphyria cutanea tarda, blooddisorders, such as β-thalassemia or sickle cell disease, congenitaldyserythropoietic anemia, marrow failure syndromes, myelodysplasia,transfusional iron overload, a disorder associated with iron overload,Parkinson's Disease, Alzheimer's Disease or Friedreich's Ataxiaassociated with iron overload, and infections and non-relapse relatedmortality associated with bone marrow transplantation, preferablyhemochromatosis, wherein the nucleic acid inhibits expression ofTMPRSS6, wherein the nucleic acid comprises at least one duplex regionthat comprises at least a portion of a first strand and at least aportion of a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of RNA transcribed from the TMPRSS6gene, wherein the first strand comprises the nucleotide sequence of SEQID NO:17 and the second strand comprises the nucleotide sequence of SEQID NO:18, and wherein the nucleic acid is administered with deferiprone.14. The combination, combination for use, nucleic acid for use, kit,composition or composition for use according to any preceding claim,wherein the nucleic acid and one or more iron chelators are administeredsimultaneously, separately or sequentially.
 15. The combination,combination for use, nucleic acid for use, kit, composition orcomposition for use according to any preceding claim, wherein thenucleic acid is conjugated to a ligand, optionally at the 5′ end of thesecond strand, optionally wherein the ligand comprises (i) one or moreN-acetyl galactosamine (GalNAc) moieties and derivatives thereof, and(ii) a linker, wherein the linker conjugates the GalNAc moieties to thenucleic acid.
 16. The combination, combination for use, nucleic acid foruse, kit, composition or composition for use according to claim 15,wherein the conjugated nucleic acid has the structure:

wherein Z is a nucleic acid according to any of claims 1 to
 14. 17. Acombination, combination for use, or nucleic acid for use according toany one of claims 1 to 14, or a conjugated nucleic acid according toclaims 15-16, wherein the nucleic acid is stabilised at the 5′ and/or 3′end of either or both strands, optionally wherein the nucleic acidcomprises two phosphorothioate linkages between each of the threeterminal 3′ and between each of the three terminal 5′ nucleotides on thefirst strand, and two phosphorothioate linkages between the threeterminal nucleotides of the 3′ end of the second strand, having thestructure 5′-3′ TMPRSS6-hcm-9A 6 (ps) 2 (ps) 736462826472845 (ps) 4 (ps)6 5′-3′ TMPRSS6-hcm-9B 17273547153517184 (ps) 5 (ps) 1

wherein the specific modifications are depicted by the following numbers1=2′F-dU, 2=2′F-dA, 3=2′F-dC, 4=2′F-dG, 5=2′-OMe-rU; 6=2′-OMe-rA;7=2′-OMe-rC; 8=2′-OMe-rG (ps)=phosphorothioate linkage.
 18. A method oftreating a disease or disorder comprising administration of acombination comprising a nucleic acid or conjugated nucleic acid and oneor more iron chelators according to any preceding claim to an individualin need of treatment thereof.